Method and Compositions for Synthesizing Improved Silk Fibers

ABSTRACT

The present disclosure provides methods and compositions for directed to synthetic block copolymer proteins, expression constructs for their secretion, recombinant microorganisms for their production, and synthetic fibers (including advantageously, microfibers) comprising these proteins that recapitulate many properties of natural silk. The recombinant microorganisms can be used for the commercial production of silk-like fibers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending International Application No. PCT/US2014/056117, filed Sep. 17, 2014, which claims benefit of U.S. Provisional Application No. 61/878,858, filed on Sep. 17, 2013, both of which are hereby incorporated by reference in their entirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 16, 2014, is named 27321PCT_CRF_SequenceListing.txt and is 4,189,730 bytes in size.

FIELD OF THE INVENTION

The present disclosure relates to methods and compositions directed to synthetic block copolymer proteins, expression constructs for their secretion, recombinant microorganisms for their production, and synthetic fibers comprising these proteins that recapitulate many properties of natural silk.

BACKGROUND OF THE INVENTION

Spider's silk polypeptides are large (>150 kDa, >1000 amino acids) polypeptides that can be broken down into three domains: an N-terminal non-repetitive domain (NTD), the repeat domain (REP), and the C-terminal non-repetitive domain (CTD). The NTD and CTD are relatively small (˜150, ˜100 amino acids respectively), well-studied, and are believed to confer to the polypeptide aqueous stability, pH sensitivity, and molecular alignment upon aggregation. NTD also has a strongly predicted secretion tag, which is often removed during heterologous expression. The repetitive region composes ˜90% of the natural polypeptide, and folds into the crystalline and amorphous regions that confer strength and flexibility to the silk fiber, respectively.

Silk polypeptides come from a variety of sources, including bees, moths, spiders, mites, and other arthropods. Some organisms make multiple silk fibers with unique sequences, structural elements, and mechanical properties. For example, orb weaving spiders have six unique types of glands that produce different silk polypeptide sequences that are polymerized into fibers tailored to fit an environmental or lifecycle niche. The fibers are named for the gland they originate from and the polypeptides are labeled with the gland abbreviation (e.g. “Ma”) and “Sp” for spidroin (short for spider fibroin). In orb weavers, these types include Major Ampullate (MaSp, also called dragline), Minor Ampullate (MiSp), Flagelliform (Flag), Aciniform (AcSp), Tubuliform (TuSp), and Pyriform (PySp). This combination of polypeptide sequences across fiber types, domains, and variation amongst different genus and species of organisms leads to a vast array of potential properties that can be harnessed by commercial production of the recombinant fibers. To date, the vast majority of the work with recombinant silks has focused on the Major Ampullate Spidroins (MaSp).

Currently, recombinant silk fibers are not commercially available and, with a handful of exceptions, are not produced in microorganisms outside of Escherichia coli and other gram-negative prokaryotes. Recombinant silks produced to date have largely consisted either of polymerized short silk sequence motifs or fragments of native repeat domains, sometimes in combination with NTDs and/or CTDs. This has resulted in the production of small scales of recombinant silk polypeptides (milligrams at lab scale, kilograms at bioprocessing scale) produced using intracellular expression and purification by chromatography or bulk precipitation. These methods do not lead to viable commercial scalability that can compete with the price of existing technical and textile fibers. Additional production hosts that have been utilized to make silk polypeptides include transgenic goats, transgenic silkworms, and plants. These hosts have yet to enable commercial scale production of silk, presumably due to slow engineering cycles and poor scalability.

Microfibers are a classification of fibers having a fineness of less than 1 decitex (dtex), approximately 10 μm in diameter. H. K., Kaynak and O. Babaarslan, Woven Fabrics, Croatia: InTech, 2012. The small diameter of microfibers imparts a range of qualities and characteristics to microfiber yarns and fabrics that are desirable to consumers. Microfibers are inherently more flexible (bending is inversely proportional to fiber diameter) and thus have a soft feel, low stiffness, and high drapeability. Microfibers can also be spun into yarns having high fiber density (greater fibers per yarn cross-sectional area), giving microfiber yarns a higher strength compared to other yarns of similar dimensions. Microfibers also contribute to discrete stress relief within the yarn, resulting in anti-wrinkle fabrics. Furthermore, microfibers have high compaction efficiency within the yarn, which improves fabric waterproofness and windproofness while maintaining breathability compared to other waterproofing and windproofing techniques (such as polyvinyl coatings). The high density of fibers within microfiber fabrics results in microchannel structures between fibers, which promotes the capillary effect and imparts a wicking and quick drying characteristic. The high surface area to volume of microfiber yarns allows for brighter and sharper dyeing, and printed fabrics have clearer and sharper pattern retention as well. Currently, recombinant silk fibers do not have a fineness that is small enough to result in silks having microfiber type characteristics. U.S. Pat. App. Pub. No. 2014/0058066 generally discloses fiber diameters between 5-100 μm, but does not actually disclose any working examples of any fiber having a diameter as small as 5 μm.

What is needed, therefore, are improved methods and compositions relating to of recombinant block copolymer proteins, expression constructs for their secretion at high rates, microorganisms expressing these proteins and synthetic fibers made from these proteins that recapitulate many of the properties of silk fibers, including fibers having small diameters useful for microfiber textiles.

SUMMARY OF THE INVENTION

The invention provides compositions of proteinaceous block co-polymers capable of assembling into fibers, and methods of producing said co-polymers. A proteinaceous block co-polymer comprises a quasi-repeat domain, the co-polymer capable of assembling into a fiber. In some embodiments the co-polymer comprises an alanine composition of 12-40% of the amino acid sequence of the co-polymer, a glycine composition of 25-50% of the amino acid sequence of the co-polymer, a proline composition of 9-20% of the amino acid sequence of the co-polymer, a β-turn composition of 15-37% of the amino acid sequence of the co-polymer, a GPG amino acid motif content of 18-55% of the amino acid sequence of the co-polymer, and a poly alanine amino acid motif content of 9-35% of all amino acids of the co-polymer.

In some embodiments, the co-polymer also includes an N-terminal non-repetitive domain between 75-350 amino acids in length, and a C-terminal non-repetitive domain between 75-350 amino acids in length. In some embodiments, the quasi-repeat domain is 500-5000, 119-1575, or 900-950 amino acids in length. In other embodiments, the mass of the co-polymer is 40-400, 12.2-132, or 70-100 kDa. In some embodiments, the alanine composition is 16-31% or 15-20% of the amino acid sequence of the co-polymer. In other embodiments, the glycine composition is 29-43% or 38-43% of the amino acid sequence of the co-polymer. In some embodiments, the proline composition is 11-16% or 13-15% of the amino acid sequence of the co-polymer. In other embodiments, the β-turn composition is 18-33% or 25-30% of the amino acid sequence of the co-polymer. In some embodiments, the GPG amino acid motif content is 22-47% or 30-45% of the amino acid sequence of the co-polymer. In other embodiments, the poly alanine amino acid motif content is 12-29% of the amino acid sequence of the co-polymer. In some embodiments, the co-polymer comprises a sequence from Table 13a, SEQ ID NO: 1396, or SEQ ID NO: 1374. In other embodiments, the co-polymer consists of SEQ ID NO: 1398 or SEQ ID NO: 2770.

In some embodiments, an engineered microorganism comprises a heterologous nucleic acid molecule encoding a secretion signal and a coding sequence, the coding sequence encoding the co-polymer described above, wherein the secretion signal allows for secretion of the co-polymer from the microorganism. In further embodiments, the engineered microorganism is Pichia pastoris or Bacillus subtilis. In other embodiments, a cell culture comprises a culture medium and the engineered microorganism. In other embodiments, a method of producing a secreted block co-polymer comprises obtaining the cell culture medium and maintaining the cell culture medium under conditions that result in the engineered microorganism secreting the co-polymer at a rate of at least 2-20 mg silk/g DCW/hour. In further embodiments, the co-polymer is secreted at a rate of at least 20 mg silk/g DCW/hour. In yet other embodiments, a cell culture medium comprises a secreted co-polymer as described above.

In other embodiments, the invention includes a method for producing a fiber comprises obtaining the cell culture medium as described above, isolating the secreted protein, and processing the protein into a spinnable solution and producing a fiber from the spinnable solution. In some embodiments, a fiber comprises a secreted co-polymer as described above. In some embodiments, the fiber has a yield stress of 24-172 or 150-172 MPa. In other embodiments, the fiber has a maximum stress of 54-310 or 150-310 MPa. In some embodiments, the fiber has a breaking strain of 2-200% or 180-200%. In other embodiments, the fiber has a diameter of 4.48-12.7 or 4-5 μm. In some embodiments, the fiber has an initial modulus of 1617-5820 or 5500-5820 MPa. In other embodiments, the fiber has a toughness value of at least 0.5, 3.1, or 59.2 MJ/m³. In still other embodiments, the fiber has a fineness between 0.2-0.6 denier.

These and other embodiments of the invention are further described in the Figures, Description, Examples and Claims, herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the hierarchical architecture of silk polypeptide sequences, including the block copolymeric structure of natural silk polypeptides. FIG. 1 discloses “AAAAAA” as SEQ ID NO: 2838.

FIG. 2 shows a screening process for silk polypeptide domains and their DNA encoding according to some embodiments of the invention.

FIG. 3 shows how silk repeat sequences and terminal domains that pass preliminary screening are assembled to create functional block copolymers that can be purified and made into fibers, according to an embodiment of the invention.

FIG. 4 shows a representative western blot of expressed silk repeat sequences and terminal domain sequences.

FIG. 5 shows a representative western blot of expressed silk repeat sequences and terminal domain sequences.

FIG. 6 depicts assembly of a block copolymer 18B silk polynucleotide from repeat sequences R1, R2, according to an embodiment of the invention.

FIG. 7 depicts assembly vectors used to assemble silk polynucleotide segments, according to an embodiment of the invention.

FIG. 8 shows ligation of 2 sequences to form a part of a silk polynucleotide sequence, according to an embodiment of the invention. FIG. 8 discloses SEQ ID NOs: 2839-2842 and 2841-2843, respectively, in order of appearance.

FIG. 9 is a western blot comprising block copolymer silk polypeptides isolated from a culture expressing an 18B silk polypeptide.

FIG. 10 is a light microscopy magnified view of a block copolymer fiber produced by methods described herein.

FIG. 11 shows a graph of stress v. strain for several block copolymer fibers produced according to methods described herein.

FIG. 12 is an assembly diagram of several silk R domains to form a block copolymer polynucleotide, according to an embodiment of the invention.

FIG. 13 shows a western blot of expressed block copolymer polypeptides each polypeptide being a concatamer of four copies of the indicated silk repeat sequences.

FIG. 14 shows representative western blots of additional expressed block copolymer polypeptides built using silk repeat sequences and expressed silk terminal domain sequences.

FIG. 15 illustrates the assembly of circularly permuted variants of an 18B polypeptide, according to embodiments of the invention.

FIG. 16 shows a western blot of expressed block copolymer peptides build using silk repeat domains consisting of between 1 and 6 R domains, including circularly permuted variants and variants expressed by different promoters or different copy numbers.

FIG. 17 are stress-strain curves showing the effect of draw ratio of block copolymer fibers of an 18B polypeptide.

FIG. 18 is a stress-strain curve for a block copolymer fiber comprising SEQ ID NO: 1398.

FIG. 19 shows the results of FTIR spectra for untreated and annealed block copolymer fibers.

FIG. 20 shows scanning electron micrographs of block copolymer fibers of the invention.

FIG. 21 illustrates graphs showing the amino acid content of various silk repeat sequences that can be expressed as block copolymers useful for the production of fibers.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and polypeptide and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.

The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; Handbook of Biochemistry: Section A Proteins, Vol I, CRC Press (1976); Handbook of Biochemistry: Section A Proteins, Vol II, CRC Press (1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).

All publications, patents and other references mentioned herein are hereby incorporated by reference in their entireties.

The following terms, unless otherwise indicated, shall be understood to have the following meanings:

The term “polynucleotide” or “nucleic acid molecule” refers to a polymeric form of nucleotides of at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native internucleoside bonds, or both. The nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation.

Unless otherwise indicated, and as an example for all sequences described herein under the general format “SEQ ID NO:”, “nucleic acid comprising SEQ ID NO:1” refers to a nucleic acid, at least a portion of which has either (i) the sequence of SEQ ID NO:1, or (ii) a sequence complementary to SEQ ID NO:1. The choice between the two is dictated by the context. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target.

An “isolated” RNA, DNA or a mixed polymer is one which is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, e.g., ribosomes, polymerases and genomic sequences with which it is naturally associated.

The term “recombinant” refers to a biomolecule, e.g., a gene or polypeptide, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term “recombinant” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as polypeptides and/or mRNAs encoded by such nucleic acids.

As used herein, an endogenous nucleic acid sequence in the genome of an organism (or the encoded polypeptide product of that sequence) is deemed “recombinant” herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered. In this context, a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous (originating from the same host cell or progeny thereof) or exogenous (originating from a different host cell or progeny thereof). By way of example, a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell, such that this gene has an altered expression pattern. This gene would now become “recombinant” because it is separated from at least some of the sequences that naturally flank it. In an embodiment, a heterologous nucleic acid molecule is not endogenous to the organism. In further embodiments, a heterologous nucleic acid molecule is a plasmid or molecule integrated into a host chromosome by homologous or random integration.

A nucleic acid is also considered “recombinant” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome. For instance, an endogenous coding sequence is considered “recombinant” if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention. A “recombinant nucleic acid” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.

As used herein, the phrase “degenerate variant” of a reference nucleic acid sequence encompasses nucleic acid sequences that can be translated, according to the standard genetic code, to provide an amino acid sequence identical to that translated from the reference nucleic acid sequence. The term “degenerate oligonucleotide” or “degenerate primer” is used to signify an oligonucleotide capable of hybridizing with target nucleic acid sequences that are not necessarily identical in sequence but that are homologous to one another within one or more particular segments.

The term “percent sequence identity” or “identical” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990) (hereby incorporated by reference in its entirety). For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference. Alternatively, sequences can be compared using the computer program, BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

The term “substantial homology” or “substantial similarity,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 76%, 80%, 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.

The nucleic acids (also referred to as polynucleotides) of this present invention can include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They can be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as the modifications found in “locked” nucleic acids.

The term “mutated” when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence may be inserted, deleted or changed compared to a reference nucleic acid sequence. A single alteration may be made at a locus (a point mutation) or multiple nucleotides may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence. A nucleic acid sequence may be mutated by any method known in the art including but not limited to mutagenesis techniques such as “error-prone PCR” (a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product; see, e.g., Leung et al., Technique, 1:11-15 (1989) and Caldwell and Joyce, PCR Methods Applic. 2:28-33 (1992)); and “oligonucleotide-directed mutagenesis” (a process which enables the generation of site-specific mutations in any cloned DNA segment of interest; see, e.g., Reidhaar-Olson and Sauer, Science 241:53-57 (1988)).

The term “vector” as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which generally refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, but also includes linear double-stranded molecules such as those resulting from amplification by the polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme. Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC). Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome (discussed in more detail below). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain preferred vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”).

The term “expression system” as used herein includes vehicles or vectors for the expression of a gene in a host cell as well as vehicles or vectors which bring about stable integration of a gene into the host chromosome.

“Operatively linked” or “operably linked” expression control sequences refers to a linkage in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest.

The term “expression control sequence” as used herein refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance polypeptide stability; and when desired, sequences that enhance polypeptide secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

The term “promoter,” as used herein, refers to a DNA region to which RNA polymerase binds to initiate gene transcription, and positions at the 5′ direction of an mRNA transcription initiation site.

The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism.

The term “peptide” as used herein refers to a short polypeptide, e.g., one that is typically less than about 50 amino acids long and more typically less than about 30 amino acids long. The term as used herein encompasses analogs and mimetics that mimic structural and thus biological function.

The term “polypeptide” encompasses both naturally-occurring and non-naturally-occurring proteins, and fragments, mutants, derivatives and analogs thereof. A polypeptide may be monomeric or polymeric. Further, a polypeptide may comprise a number of different domains each of which has one or more distinct activities.

As used herein, the term “molecule” means any compound, including, but not limited to, a small molecule, peptide, polypeptide, sugar, nucleotide, nucleic acid, polynucleotide, lipid, etc., and such a compound can be natural or synthetic.

The term “block” or “repeat unit” as used herein refers to a subsequence greater than approximately 12 amino acids of a natural silk polypeptide that is found, possibly with modest variations, repeatedly in said natural silk polypeptide sequence and serves as a basic repeating unit in said silk polypeptide sequence. Examples can be found in Table 1. Further examples of block amino acid sequences can be found in SEQ ID NOs: 1515-2156. Blocks may, but do not necessarily, include very short “motifs.” A “motif” as used herein refers to an approximately 2-10 amino acid sequence that appears in multiple blocks. For example, a motif may consist of the amino acid sequence GGA, GPG, or AAAAA (SEQ ID NO: 2803). A sequence of a plurality of blocks is a “block co-polymer.”

As used herein, the term “repeat domain” refers to a sequence selected from the set of contiguous (unbroken by a substantial non-repetitive domain, excluding known silk spacer elements) repetitive segments in a silk polypeptide. Native silk sequences generally contain one repeat domain. In some embodiments of the present invention, there is one repeat domain per silk molecule. A “macro-repeat” as used herein is a naturally occurring repetitive amino acid sequence comprising more than one block. In an embodiment, a macro-repeat is repeated at least twice in a repeat domain. In a further embodiment, the two repetitions are imperfect. A “quasi-repeat” as used herein is an amino acid sequence comprising more than one block, such that the blocks are similar but not identical in amino acid sequence.

A “repeat sequence” or “R” as used herein refers to a repetitive amino acid sequence. Examples include the nucleotide sequences of SEQ ID NOs: 1-467, the nucleotide sequences with flanking sequences for cloning of SEQ ID NOs: 468-931, and the amino acid sequences of SEQ ID NOs: 932-1398. In an embodiment, a repeat sequence includes a macro-repeat or a fragment of a macro-repeat. In another embodiment, a repeat sequence includes a block. In a further embodiment, a single block is split across two repeat sequences.

Any ranges disclosed herein are inclusive of the extremes of the range. For example, a range of 2-5% includes 2% and 5%, and any number or fraction of a number in between, for example: 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, and 4.75%.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this present invention pertains. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention and will be apparent to those of skill in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

Throughout this specification and claims, the word “comprise” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Silk Sequences

In some embodiments disclosed herein are 1) block copolymer polypeptide compositions generated by mixing and matching repeat domains derived from silk polypeptide sequences and 2) recombinant expression of block copolymer polypeptides having sufficiently large size (approximately 40 kDa) to form useful fibers by secretion from an industrially scalable microorganism. We provide herein the ability to produce relatively large (approximately 40 kDa to approximately 100 kDa) block copolymer polypeptides engineered from silk repeat domain fragments in a scalable engineered microorganism host, including sequences from almost all published amino acid sequences of spider silk polypeptides. In some embodiments, silk polypeptide sequences are matched and designed to produce highly expressed and secreted polypeptides capable of fiber formation.

Provided herein, in several embodiments, are compositions for expression and secretion of block copolymers engineered from a combinatorial mix of silk polypeptide domains across the silk polypeptide sequence space. In some embodiments provided herein are methods of secreting block copolymers in scalable organisms (e.g., yeast, fungi, and gram positive bacteria). In some embodiments, the block copolymer polypeptide comprises 0 or more N-terminal domains (NTD), 1 or more repeat domains (REP), and 0 or more C-terminal domains (CTD). In some aspects of the embodiment, the block copolymer polypeptide is >100 amino acids of a single polypeptide chain. In some embodiments, the block copolymer polypeptide comprises a domain that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence of SEQ ID NOs: 932-1398.

Several types of native spider silks have been identified. The mechanical properties of each natively spun silk type are believed to be closely connected to the molecular composition of that silk. See, e.g., Garb, J. E., et al., Untangling spider silk evolution with spidroin terminal domains, BMC Evol. Biol., 10:243 (2010); Bittencourt, D., et al., Protein families, natural history and biotechnological aspects of spider silk, Genet. Mol. Res., 11:3 (2012); Rising, A., et al., Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications, Cell. Mol. Life Sci., 68:2, pg. 169-184 (2011); and Humenik, M., et al., Spider silk: understanding the structure-function relationship of a natural fiber, Prog. Mol. Biol. Transl. Sci., 103, pg. 131-85 (2011). For example:

Aciniform (AcSp) silks tend to have high toughness, a result of moderately high strength coupled with moderately high extensibility. AcSp silks are characterized by large block (“ensemble repeat”) sizes that often incorporate motifs of poly serine and GPX. Tubuliform (TuSp or Cylindrical) silks tend to have large diameters, with modest strength and high extensibility. TuSp silks are characterized by their poly serine and poly threonine content, and short tracts of poly alanine. Major Ampullate (MaSp) silks tend to have high strength and modest extensibility. MaSp silks can be one of two subtypes: MaSp1 and MaSp2. MaSp1 silks are generally less extensible than MaSp2 silks, and are characterized by poly alanine, GX, and GGX motifs. MaSp2 silks are characterized by poly alanine, GGX, and GPX motifs. Minor Ampullate (MiSp) silks tend to have modest strength and modest extensibility. MiSp silks are characterized by GGX, GA, and poly A motifs, and often contain spacer elements of approximately 100 amino acids. Flagelliform (Flag) silks tend to have very high extensibility and modest strength. Flag silks are usually characterized by GPG, GGX, and short spacer motifs.

The properties of each silk type can vary from species to species, and spiders leading distinct lifestyles (e.g. sedentary web spinners vs. vagabond hunters) or that are evolutionarily older may produce silks that differ in properties from the above descriptions (for descriptions of spider diversity and classification, see Hormiga, G., and Griswold, C. E., Systematics, phylogeny, and evolution of orb-weaving spiders, Annu. Rev. Entomol. 59, pg. 487-512 (2014); and Blackedge, T. A. et al., Reconstructing web evolution and spider diversification in the molecular era, Proc. Natl. Acad. Sci. U.S.A., 106:13, pg. 5229-5234 (2009)). However, synthetic block copolymer polypeptides having sequence similarity and/or amino acid composition similarity to the repeat domains of native silk proteins can be used to manufacture on commercial scales consistent silk-like fibers that recapitulate the properties of corresponding natural silk fibers.

In some embodiments, a list of putative silk sequences can be compiled by searching GenBank for relevant terms, e.g. “spidroin” “fibroin” “MaSp”, and those sequences can be pooled with additional sequences obtained through independent sequencing efforts. Sequences are then translated into amino acids, filtered for duplicate entries, and manually split into domains (NTD, REP, CTD). In some embodiments, candidate amino acid sequences are reverse translated into a DNA sequence optimized for expression in Pichia (Komagataella) pastoris. The DNA sequences are each cloned into an expression vector and transformed into Pichia (Komagataella) pastoris. In some embodiments, various silk domains demonstrating successful expression and secretion are subsequently assembled in combinatorial fashion to build silk molecules capable of fiber formation.

Silk polypeptides are characteristically composed of a repeat domain (REP) flanked by non-repetitive regions (e.g., C-terminal and N-terminal domains). In an embodiment, both the C-terminal and N-terminal domains are between 75-350 amino acids in length. The repeat domain exhibits a hierarchical architecture, as depicted in FIG. 1. The repeat domain comprises a series of blocks (also called repeat units). The blocks are repeated, sometimes perfectly and sometimes imperfectly (making up a quasi-repeat domain), throughout the silk repeat domain. The length and composition of blocks varies among different silk types and across different species. Table 1 lists examples of block sequences from selected species and silk types, with further examples presented in Rising, A. et al., Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications, Cell Mol. Life Sci., 68:2, pg 169-184 (2011); and Gatesy, J. et al., Extreme diversity, conservation, and convergence of spider silk fibroin sequences, Science, 291:5513, pg. 2603-2605 (2001). In some cases, blocks may be arranged in a regular pattern, forming larger macro-repeats that appear multiple times (usually 2-8) in the repeat domain of the silk sequence. Repeated blocks inside a repeat domain or macro-repeat, and repeated macro-repeats within the repeat domain, may be separated by spacing elements. In some embodiments, block sequences comprise a glycine rich region followed by a polyA region. In some embodiments, short (˜1-10) amino acid motifs appear multiple times inside of blocks. A subset of commonly observed motifs is depicted in FIG. 1. For the purpose of this invention, blocks from different natural silk polypeptides can be selected without reference to circular permutation (i.e., identified blocks that are otherwise similar between silk polypeptides may not align due to circular permutation). Thus, for example, a “block” of SGAGG (SEQ ID NO: 2804) is, for the purposes of the present invention, the same as GSGAG (SEQ ID NO: 2805) and the same as GGSGA (SEQ ID NO: 2806); they are all just circular permutations of each other. The particular permutation selected for a given silk sequence can be dictated by convenience (usually starting with a G) more than anything else. Silk sequences obtained from the NCBI database can be partitioned into blocks and non-repetitive regions.

TABLE 1 Samples of Block Sequences Silk Species Type Representative Block Amino Acid Sequence Aliatypus Fibroin GAASSSSTIITTKSASASAAADASAAATASAASRSSAN gulosus 1 AAASAFAQSFSSILLESGYFCSIFGSSISSSYAAAIASAA SRAAAESNGYTTHAYACAKAVASAVERVTSGADAY AYAQAISDALSHALLYTGRLNTANANSLASAFAYAF ANAAAQASASSASAGAASASGAASASGAGSAS  (SEQ ID NO: 2807) Plectreurys Fibroin GAGAGAGAGAGAGAGAGSGASTSVSTSSSSGSGAGA tristis 1 GAGSGAGSGAGAGSGAGAGAGAGGAGAGFGSGLGL GYGVGLSSAQAQAQAQAAAQAQAQAQAQAYAAAQ AQAQAQAQAQAAAAAAAAAAA (SEQ ID NO: 2808) Plectreurys Fibroin GAAQKQPSGESSVATASAAATSVTSGGAPVGKPGVP tristis 4 APIFYPQGPLQQGPAPGPSNVQPGTSQQGPIGGVGGS NAFSSSFASALSLNRGFTEVISSASATAVASAFQKGLA PYGTAFALSAASAAADAYNSIGSGANAFAYAQAFAR VLYPLVQQYGLSSSAKASAFASAIASSFSSGTSGQGPS IGQQQPPVTISAASASAGASAAAVGGGQVGQGPYGG QQQSTAASASAAAATATS (SEQ ID NO: 2809) Araneus TuSp GNVGYQLGLKVANSLGLGNAQALASSLSQAVSAVG gemmoides VGASSNAYANAVSNAVGQVLAGQGILNAANAGSLA SSFASALSSSAASVASQSASQSQAASQSQAAASAFRQ AASQSASQSDSRAGSQSSTKTTSTSTSGSQADSRSASS SASQASASAFAQQSSASLSSSSSFSSAFSSATSISAV (SEQ ID NO: 2810) Argiope TuSp GSLASSFASALSASAASVASSAAAQAASQSQAAASAF aurantia SRAASQSASQSAARSGAQSISTTTTTSTAGSQAASQSA SSAASQASASSFARASSASLAASSSFSSAFSSANSLSAL GNVGYQLGFNVANNLGIGNAAGLGNALSQAVSSVG VGASSSTYANAVSNAVGQFLAGQGILNAANA  (SEQ ID NO: 2811) Deinopis TuSp GASASAYASAISNAVGPYLYGLGLFNQANAASFASSF spinosa ASAVSSAVASASASAASSAYAQSAAAQAQAASSAFS QAAAQSAAAASAGASAGAGASAGAGAVAGAGAVA GAGAVAGASAAAASQAAASSSASAVASAFAQSASY ALASSSAFANAFASATSAGYLGSLAYQLGLTTAYNL GLSNAQAFASTLSQAVTGVGL (SEQ ID NO: 2812) Nephila TuSp GATAASYGNALSTAAAQFFATAGLLNAGNASALASS clavipes FARAFSASAESQSFAQSQAFQQASAFQQAASRSASQS AAEAGSTSSSTTTTTSAARSQAASQSASSSYSSAFAQA ASSSLATSSALSRAFSSVSSASAASSLAYSIGLSAARSL GIADAAGLAGVLARAAGALGQ (SEQ ID NO: 2813) Argiope Flag GGAPGGGPGGAGPGGAGFGPGGGAGFGPGGGAGFG trifasciata PGGAAGGPGGPGGPGGPGGAGGYGPGGAGGYGPGG VGPGGAGGYGPGGAGGYGPGGSGPGGAGPGGAGGE GPVTVDVDVTVGPEGVGGGPGGAGPGGAGFGPGGG AGFGPGGAPGAPGGPGGPGGPGGPGGPGGVGPGGA GGYGPGGAGGVGPAGTGGFGPGGAGGFGPGGAGGF GPGGAGGFGPAGAGGYGPGGVGPGGAGGFGPGGVG PGGSGPGGAGGEGPVTVDVDVSV (SEQ ID NO: 2814) Nephila Flag GVSYGPGGAGGPYGPGGPYGPGGEGPGGAGGPYGP clavipes GGVGPGGSGPGGYGPGGAGPGGYGPGGSGPGGYGP GGSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGYGP GGSGPGGSGPGGSGPGGYGPGGTGPGGSGPGGYGPG GSGPGGSGPGGYGPGGSGPGGFGPGGSGPGGYGPGG SGPGGAGPGGVGPGGFGPGGAGPGGAAPGGAGPGG AGPGGAGPGGAGPGGAGPGGAGPGGAGGAGGAGGS GGAGGSGGTTIIEDLDITIDGADGPITISEELPISGAGGS GPGGAGPGGVGPGGSGPGGVGPGGSGPGGVGPGGS GPGGVGPGGAGGPYGPGGSGPGGAGGAGGPGGAYG PGGSYGPGGSGGPGGAGGPYGPGGEGPGGAGGPYGP GGAGGPYGPGGAGGPYGPGGEGGPYGP  (SEQ ID NO: 2815) Latrodectus AcSp GINVDSDIGSVTSLILSGSTLQMTIPAGGDDLSGGYPG hesperus GFPAGAQPSGGAPVDFGGPSAGGDVAAKLARSLAST LASSGVFRAAFNSRVSTPVAVQLTDALVQKIASNLGL DYATASKLRKASQAVSKVRMGSDTNAYALAISSALA EVLSSSGKVADANINQIAPQLASGIVLGVSTTAPQFGV DLSSINVNLDISNVARNMQASIQGGPAPITAEGPDFGA GYPGGAPTDLSGLDMGAPSDGSRGGDATAKLLQAL VPALLKSDVFRAIYKRGTRKQVVQYVTNSALQQAAS SLGLDASTISQLQTKATQALSSVSADSDSTAYAKAFG LAIAQVLGTSGQVNDANVNQIGAKLATGILRGSSAV APRLGIDLS (SEQ ID NO: 2816) Argiope AcSp GAGYTGPSGPSTGPSGYPGPLGGGAPFGQSGFGGSAG trifasciata PQGGFGATGGASAGLISRVANALANTSTLRTVLRTG VSQQIASSVVQRAAQSLASTLGVDGNNLARFAVQAV SRLPAGSDTSAYAQAFSSALFNAGVLNASNIDTLGSR VLSALLNGVSSAAQGLGINVDSGSVQSDISSSSSFLST SSSSASYSQASASSTS (SEQ ID NO: 2817) Uloborus AcSp GASAADIATAIAASVATSLQSNGVLTASNVSQLSNQL diversus ASYVSSGLSSTASSLGIQLGASLGAGFGASAGLSASTD ISSSVEATSASTLSSSASSTSVVSSINAQLVPALAQTAV LNAAFSNINTQNAIRIAELLTQQVGRQYGLSGSDVAT ASSQIRSALYSVQQGSASSAYVSAIVGPLITALSSRGV VNASNSSQIASSLATAILQFTANVAPQFGISIPTSAVQS DLSTISQSLTAISSQTSSSVDSSTSAFGGISGPSGPSPYG PQPSGPTFGPGPSLSGLTGFTATFASSFKSTLASSTQFQ LIAQSNLDVQTRSSLISKVLINALSSLGISASVASSIAAS SSQSLLSVSA (SEQ ID NO: 2818) Euprosthenops  MaSp1 GGQGGQGQGRYGQGAGSSAAAAAAAAAAAAAA australis (SEQ ID NO: 2819) Tetragnatha MaSp1 GGLGGGQGAGQGGQQGAGQGGYGSGLGGAGQGAS kauaiensis AAAAAAAA (SEQ ID NO: 2820) Argiope MaSp2 GGYGPGAGQQGPGSQGPGSGGQQGPGGLGPYGPSA aurantia AAAAAAA (SEQ ID NO: 2821) Deinopis MaSp2 GPGGYGGPGQQGPGQGQYGPGTGQQGQGPSGQQGP spinosa AGAAAAAAAAA (SEQ ID NO: 2822) Nephila MaSp2 GPGGYGLGQQGPGQQGPGQQGPAGYGPSGLSGPGG clavata AAAAAAA (SEQ ID NO: 2823)

The construction of fiber-forming block copolymer polypeptides from the blocks and/or macro-repeat domains, according to certain embodiments of the invention, is shown in FIGS. 2 and 3. FIG. 2 illustrates the division of silk sequences into distinct domains. Natural silk sequences 200 obtained from a protein database such as GenBank or through de novo sequencing are broken up by domain (N-terminal domain 202, repeat domain 204, and C-terminal domain 206). The N-terminal domain 202 and C-terminal domain 206 sequences selected for the purpose of synthesis and assembly into fibers include natural amino acid sequence information and other modifications described herein. The repeat domain 204 is decomposed into repeat sequences 208 containing representative blocks, usually 1-8 depending upon the type of silk, that capture critical amino acid information while reducing the size of the DNA encoding the amino acids into a readily synthesizable fragment. FIG. 3 illustrates how select NT 202, CT 206, and repeat sequences 208 can be assembled to create block copolymer polypeptides that can be purified and made into fibers that recapitulate the functional properties of silk, according to an embodiment of the invention. Individual NT, CT, and repeat sequences that have been verified to express and secrete are assembled into functional block copolymer polypeptides. In some embodiments, a properly formed block copolymer polypeptide comprises at least one repeat domain comprising at least 1 repeat sequence 208, and is optionally flanked by an N-terminal domain 202 and/or a C-terminal domain 206.

In some embodiments, a repeat domain comprises at least one repeat sequence. In some embodiments, the repeat sequence, N-terminal domain sequence, and/or C-terminal domain sequence is selected from SEQ ID NOs: 932-1398. In some embodiments, the repeat sequence is 150-300 amino acid residues. In some embodiments, the repeat sequence comprises a plurality of blocks. In some embodiments, the repeat sequence comprises a plurality of macro-repeats. In some embodiments, a block or a macro-repeat is split across multiple repeat sequences.

In some embodiments, the repeat sequence starts with a Glycine, and cannot end with phenylalanine (F), tyrosine (Y), tryptophan (W), cysteine (C), histidine (H), asparagine (N), methionine (M), or aspartic acid (D) to satisfy DNA assembly requirements. In some embodiments, some of the repeat sequences can be altered as compared to native sequences. In some embodiments, the repeat sequences can be altered such as by addition of a serine to the C terminus of the polypeptide (to avoid terminating in F, Y, W, C, H, N, M, or D). In some embodiments, the repeat sequence can be modified by filling in an incomplete block with homologous sequence from another block. In some embodiments, the repeat sequence can be modified by rearranging the order of blocks or macrorepeats.

In some embodiments, non-repetitive N- and C-terminal domains can be selected for synthesis (See SEQ ID NOs: 1-145). In some embodiments, N-terminal domains can be by removal of the leading signal sequence, e.g., as identified by SignalP (Peterson, T. N., et. Al., SignalP 4.0: discriminating signal peptides from transmembrane regions, Nat. Methods, 8:10, pg. 785-786 (2011).

In some embodiments, the N-terminal domain, repeat sequence, or C-terminal domain sequences can be derived from Agelenopsis aperta, Aliatypus gulosus, Aphonopelma seemanni, Aptostichus sp. AS217, Aptostichus sp. AS220, Araneus diadematus, Araneus gemmoides, Araneus ventricosus, Argiope amoena, Argiope argentata, Argiope bruennichi, Argiope trifasciata, Atypoides riversi, Avicularia juruensis, Bothriocyrtum californicum, Deinopis Spinosa, Diguetia canities, Dolomedes tenebrosus, Euagrus chisoseus, Euprosthenops australis, Gasteracantha mammosa, Hypochilus thorelli, Kukulcania hibernalis, Latrodectus hesperus, Megahexura fulva, Metepeira grandiosa, Nephila antipodiana, Nephila clavata, Nephila clavipes, Nephila madagascariensis, Nephila pilipes, Nephilengys cruentata, Parawixia bistriata, Peucetia viridans, Plectreurys tristis, Poecilotheria regalis, Tetragnatha kauaiensis, or Uloborus diversus.

In some embodiments, the silk polypeptide nucleotide coding sequence can be operatively linked to an alpha mating factor nucleotide coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence can be operatively linked to another endogenous or heterologous secretion signal coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence can be operatively linked to a 3× FLAG nucleotide coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence is operatively linked to other affinity tags such as 6-8 His residues (SEQ ID NO: 2824).

Expression Vectors

The expression vectors of the present invention can be produced following the teachings of the present specification in view of techniques known in the art. Sequences, for example vector sequences or sequences encoding transgenes, can be commercially obtained from companies such as Integrated DNA Technologies, Coralville, Iowa or DNA 2.0, Menlo Park, Calif. Exemplified herein are expression vectors that direct high-level expression of the chimeric silk polypeptides.

Another standard source for the polynucleotides used in the invention is polynucleotides isolated from an organism (e.g., bacteria), a cell, or selected tissue. Nucleic acids from the selected source can be isolated by standard procedures, which typically include successive phenol and phenol/chloroform extractions followed by ethanol precipitation. After precipitation, the polynucleotides can be treated with a restriction endonuclease which cleaves the nucleic acid molecules into fragments. Fragments of the selected size can be separated by a number of techniques, including agarose or polyacrylamide gel electrophoresis or pulse field gel electrophoresis (Care et al. (1984) Nuc. Acid Res. 12:5647-5664; Chu et al. (1986) Science 234:1582; Smith et al. (1987) Methods in Enzymology 151:461), to provide an appropriate size starting material for cloning.

Another method of obtaining the nucleotide components of the expression vectors or constructs is PCR. General procedures for PCR are taught in MacPherson et al., PCR: A PRACTICAL APPROACH, (IRL Press at Oxford University Press, (1991)). PCR conditions for each application reaction may be empirically determined. A number of parameters influence the success of a reaction. Among these parameters are annealing temperature and time, extension time, Mg2+ and ATP concentration, pH, and the relative concentration of primers, templates and deoxyribonucleotides. Exemplary primers are described below in the Examples. After amplification, the resulting fragments can be detected by agarose gel electrophoresis followed by visualization with ethidium bromide staining and ultraviolet illumination.

Another method for obtaining polynucleotides is by enzymatic digestion. For example, nucleotide sequences can be generated by digestion of appropriate vectors with suitable recognition restriction enzymes. Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTPs) using standard techniques.

Polynucleotides are inserted into suitable backbones, for example, plasmids, using methods well known in the art. For example, insert and vector DNA can be contacted, under suitable conditions, with a restriction enzyme to create complementary or blunt ends on each molecule that can pair with each other and be joined with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the termini of a polynucleotide. These synthetic linkers can contain nucleic acid sequences that correspond to a particular restriction site in the vector DNA. Other means are known and available in the art. A variety of sources can be used for the component polynucleotides.

In some embodiments, expression vectors containing an R, N, or C sequence are transformed into a host organism for expression and secretion. In some embodiments, the expression vectors comprise a secretion signal. In some embodiments, the expression vector comprises a terminator signal. In some embodiments, the expression vector is designed to integrate into a host cell genome and comprises: regions of homology to the target genome, a promoter, a secretion signal, a tag (e.g., a Flag tag), a termination/polyA signal, a selectable marker for Pichia, a selectable marker for E. coli, an origin of replication for E. coli, and restriction sites to release fragments of interest.

Host Cell Transformants

In some embodiments of the present invention, host cells transformed with the nucleic acid molecules or vectors of the present invention, and descendants thereof, are provided. In some embodiments of the present invention, these cells carry the nucleic acid sequences of the present invention on vectors, which may but need not be freely replicating vectors. In other embodiments of the present invention, the nucleic acids have been integrated into the genome of the host cells.

In some embodiments, microorganisms or host cells that enable the large-scale production of block copolymer polypeptides of the invention include a combination of: 1) the ability to produce large (>75 kDa) polypeptides, 2) the ability to secrete polypeptides outside of the cell and circumvent costly downstream intracellular purification, 3) resistance to contaminants (such as viruses and bacterial contaminations) at large-scale, and 4) the existing know-how for growing and processing the organism is large-scale (1-2000 m³) bioreactors.

A variety of host organisms can be engineered/transformed to comprise a block copolymer polypeptide expression system. Preferred organisms for expression of a recombinant silk polypeptide include yeast, fungi, and gram-positive bacteria. In certain embodiments, the host organism is Arxula adeninivorans, Aspergillus aculeatus, Aspergillus awamori, Aspergillus ficuum, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus sojae, Aspergillus tubigensis, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus anthracia, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus methanolicus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Candida boidinii, Chrysosporium lucknowense, Fusarium graminearum, Fusarium venenatum, Kluyveromyces lactis, Kluyveromyces marxianus, Myceliopthora thermophila, Neurospora crassa, Ogataea polymorpha, Penicillium camemberti, Penicillium canescens, Penicillium chrysogenum, Penicillium emersonii, Penicillium funiculosum, Penicillium griseoroseum, Penicillium purpurogenum, Penicillium roqueforti, Phanerochaete chrysosporium, Pichia angusta, Pichia methanolica, Pichia (Komagataella) pastoris, Pichia polymorpha, Pichia stipitis, Rhizomucor miehei, Rhizomucor pusillus, Rhizopus arrhizus, Streptomyces lividans, Saccharomyces cerevisiae, Schwanniomyces occidentalis, Trichoderma harzianum, Trichoderma reesei, or Yarrowia lipolytica.

In preferred aspects, the methods provide culturing host cells for direct product secretion for easy recovery without the need to extract biomass. In some embodiments, the block copolymer polypeptides are secreted directly into the medium for collection and processing.

Polypeptide Purification

The recombinant block copolymer polypeptides based on spider silk sequences produced by gene expression in a recombinant prokaryotic or eukaryotic system can be purified according to methods known in the art. In a preferred embodiment, a commercially available expression/secretion system can be used, whereby the recombinant polypeptide is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium. If expression/secretion vectors are not used, an alternative approach involves purifying the recombinant block copolymer polypeptide from cell lysates (remains of cells following disruption of cellular integrity) derived from prokaryotic or eukaryotic cells in which a polypeptide was expressed. Methods for generation of such cell lysates are known to those of skill in the art. In some embodiments, recombinant block copolymer polypeptides are isolated from cell culture supernatant.

Recombinant block copolymer polypeptide may be purified by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant polypeptide or nickel columns for isolation of recombinant polypeptides tagged with 6-8 histidine residues at their N-terminus or C-terminus Alternative tags may comprise the FLAG epitope or the hemagglutinin epitope. Such methods are commonly used by skilled practitioners.

Additionally, the method of the present invention may preferably include a purification method, comprising exposing the cell culture supernatant containing expressed block copolymer polypeptides to ammonium sulphate of 5-60% saturation, preferably 10-40% saturation.

Spinning to Generate Fibers

In some embodiments, a solution of block copolymer polypeptide of the present invention is spun into fibers using elements of processes known in the art. These processes include, for example, wet spinning, dry-jet wet spinning, and dry spinning. In preferred wetspinning embodiments, the filament is extruded through an orifice into a liquid coagulation bath. In one embodiment, the filament can be extruded through an air gap prior to contacting the coagulation bath. In a dry-jet wet spinning process, the spinning solution is attenuated and stretched in an inert, non-coagulating fluid, e.g., air, before entering the coagulating bath. Suitable coagulating fluids are the same as those used in a wetspinning process.

Preferred coagulation baths for wet spinning are maintained at temperatures of 0-90° C., more preferably 20-60° C., and are preferably about 60%, 70%, 80%, 90%, or even 100% alcohol, preferably isopropanol, ethanol, or methanol. In a preferred embodiment, the coagulation bath is 85:15% by volume methanol:water. In alternate embodiments, coagulation baths comprise ammonium sulfate, sodium chloride, sodium sulfate, or other protein precipitating salts at temperature between 20-60° C. Certain coagulant baths can be preferred depending upon the composition of the dope solution and the desired fiber properties. For example, salt based coagulant baths are preferred for an aqueous dope solution. For example, methanol is preferred to produce a circular cross section fiber. Residence times in coagulation baths can range from nearly instantaneous to several hours, with preferred residence times lasting under one minute, and more preferred residence times lasting about 20 to 30 seconds. Residence times can depend on the geometry of the extruded fiber or filament. In certain embodiments, the extruded filament or fiber is passed through more than one coagulation bath of different or same composition. Optionally, the filament or fiber is also passed through one or more rinse baths to remove residual solvent and/or coagulant. Rinse baths of decreasing salt or alcohol concentration up to, preferably, an ultimate water bath, preferably follow salt or alcohol baths.

Following extrusion, the filament or fiber can be drawn. Drawing can improve the consistency, axial orientation and toughness of the filament. Drawing can be enhanced by the composition of a coagulation bath. Drawing may also be performed in a drawing bath containing a plasticizer such as water, glycerol or a salt solution. Drawing can also be performed in a drawing bath containing a crosslinker such as gluteraldehyde or formaldehyde. Drawing can be performed at temperature from 25-100° C. to alter fiber properties, preferably at 60° C. As is common in a continuous process, drawing can be performed simultaneously during the coagulation, wash, plasticizing, and/or crosslinking procedures described previously. Drawing rates depend on the filament being processed. In one embodiment, the drawing rate is preferably about 5× the rate of reeling from the coagulation bath.

In certain embodiments of the invention, the filament is wound onto a spool after extrusion or after drawing. Winding rates are generally 1 to 500 m/min, preferably 10 to 50 m/min.

In other embodiments, to enhance the ease with which the fiber is processed, the filament can be coated with lubricants or finishes prior to winding. Suitable lubricants or finishes can be polymers or wax finishes including but not limited to mineral oil, fatty acids, isobutyl-stearate, tallow fatty acid 2-ethylhexyl ester, polyol carboxylic acid ester, coconut oil fatty acid ester of glycerol, alkoxylated glycerol, a silicone, dimethyl polysiloxane, a polyalkylene glycol, polyethylene oxide, and a propylene oxide copolymer.

The spun fibers produced by the methods of the present invention can possess a diverse range of physical properties and characteristics, dependent upon the initial properties of the source materials, i.e., the dope solution, and the coordination and selection of variable aspects of the present method practiced to achieve a desired final product, whether that product be a soft, sticky, pliable matrix conducive to cellular growth in a medical application or a load-bearing, resilient fiber, such as fishing line or cable. The tensile strength of filaments spun by the methods of the present invention generally range from 0.2 g/denier (or g/(g/9000 m)) to 3 g/denier, with filaments intended for load-bearing uses preferably demonstrating a tensile strength of at least 2 g/denier. In an embodiment, the fibers have a fineness between 0.2-0.6 denier. Such properties as elasticity and elongation at break vary dependent upon the intended use of the spun fiber, but elasticity is preferably 5% or more, and elasticity for uses in which elasticity is a critical dimension, e.g., for products capable of being “tied,” such as with sutures or laces, is preferably 10% or more. Water retention of spun fibers preferably is close to that of natural silk fibers, i.e., 10%. The diameter of spun fibers can span a broad range, dependent on the application; preferred fiber diameters range from 5, 10, 20, 30, 40, 50, 60 microns, but substantially thicker fibers may be produced, particularly for industrial applications (e.g., cable). The cross-sectional characteristics of spun fibers can vary; e.g., preferable spun fibers include circular cross-sections, elliptical, starburst cross-sections, and spun fibers featuring distinct core/sheath sections, as well as hollow fibers.

Example 1 Obtaining Silk Sequences

Silk sequences and partial sequences were obtained by searching NCBI's nucleotide database using the following terms to identify spider silks: MaSp, TuSp, CySp, MiSp, AcSp, Flag, major ampullate, minor ampullate, flagelliform, aciniform, tubuliform, cylindriform, spidroin, and spider fibroin. The resulting nucleotide sequences were translated into amino acid sequences, then curated to remove repeated sequences. Sequences that were less than 200-500 amino acids long, depending on the type of silk, were removed. Silk sequences from the above search were partitioned into blocks (e.g., repetitive sequences) and non-repetitive regions.

Repetitive polypeptide sequences (repeat (R) sequences) were selected from each silk sequence, and are listed as SEQ ID NOs: 1077-1393. Some of the R sequences have been altered, e.g., by addition of a serine to the C terminus to avoid terminating the sequence with an F, Y, W, C, H, N, M, or D amino acid. This allows for incorporation into the vector system described below. We also altered incomplete blocks by incorporation of segments from a homologous sequence from another block. For some sequences of SEQ ID NOs: 1077-1393, the order of blocks or macro-repeats has been altered from the sequence found in the NCBI database, and make up quasi-repeat domains

Non-repetitive N terminal domain sequences (N sequences) and C terminal domain sequences (C sequences) were also selected from each silk sequence (SEQ ID NOs: 932-1076). The N terminal domain sequences were altered by removal of the leading signal sequence and, if not already present, addition of an N-terminal glycine residue.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention.

Example 2 Reverse Translation of Silk Polypeptide Sequences to Nucleotide Sequences

R, N, and C amino acid sequences described in Example 1 were reverse translated to nucleotide sequences. To perform reverse translation, 10,000 candidate sequences were generated by using the Pichia (Komagataella) pastoris codon usage to bias random selection of a codon encoding the desired amino acid at each position. Select restriction sites (BsaI, BbsI, BtgZI, AscI, SbfI) were then removed from each sequence; if a site could not be removed, the sequence was discarded. Then, the entropy, longest repeated subsequence, and number of repeated 15-mers were each determined for each sequence.

To choose the optimal sequence to use for synthesis out of each set of 10,000, the following criteria were sequentially applied: keep the sequences with the lowest 25% of longest repeated subsequence, keep the sequences with the highest 10% of sequence entropy, and use the sequence with the lowest number of repeated 15-mers.

Example 3 Screening of Silk Polypeptides from Selected N, C, or R Sequences

The nucleotide sequences from Examples 1 and 2 were flanked with the following sequences during synthesis to enable cloning:

5′-GAAGACTTAGCA-SILK-GGTACGTCTTC-3′ (SEQ ID NOS 2825 and 2826) where “SILK” is a polynucleotide sequence selected according to the teachings of Example 2.

Resulting DNA was digested with BbsI and ligated into either Expression Vector RM618 (SEQ ID NO:1399) or Expression Vector RM652 (SEQ ID NO:1400) which had been digested with BtgZI and treated with Calf Intestinal Alkaline Phosphatase. Ligated material was transformed into E. coli for clonal isolation and DNA amplification using standard methods. Pichia (Komagataella) pastoris

Expression vectors containing an R, N, or C sequence were transformed into Pichia (Komagataella) pastoris (strain RMs71, which is strain GS115 (NRRL Y15851) with the mutation in the HIS4 gene restored to wild-type via transformation with a fragment of the wild-type genome (NRRLY 11430) and selection on defined medium agar plates lacking histidine) using the PEG method (Cregg, J. M., DNA-mediated transformation, Methods Mol. Biol., 389, pg. 27-42 (2007).). The expression vector consisted of a targeting region (HIS4), a dominant resistance marker (nat-conferring resistance to nourseothricin), a promoter (pGAP), a secretion signal (alpha mating factor leader and pro sequence), and a terminator (pAOX1 pA signal).

Transformants were plated on YPD agar plates containing 25 μg/ml nourseothricin and incubated for 48 hours at 30° C. Two clones from each transformation were inoculated into 400 μl of YPD in a 96-well square-well block, and incubated for 48 hours at 30° C. with agitation at 1000 rpm. Cells were pelleted via centrifugation, and the supernatant was recovered for analysis of silk polypeptide content via western blot. The resulting data demonstrates a variety of expression and secretion phenotypes, ranging from undetectable polypeptide levels in the supernatant to strong signal on the western blot indicative of relatively high titre.

Successful polypeptide expression and secretion was judged by western blot. Each western lane was scored as 1: No band 2: Moderate band or 3: Intense band. The higher of the two scores for each clone was recorded. Representative western blots with construct numbers labeled are shown in FIG. 4 and FIG. 5, with additional western blots with representative clones shown in FIG. 14. A complete listing of all R, N, and C sequences tested along with western blot results is shown in Table 2. Silk polypeptides from numerous species expressed successfully, encompassing every category of gland and all domain types.

TABLE 2 Silk polypeptide sequences Nucleotide Western Results with flanking (1 = no band Construct N/C/R Nucleotide sequences Amino acid 2 = weak band # Species sequence SEQ ID NO SEQ ID NO: SEQ ID NO: 3 = strong band) 1 Aliatypus gulosus C 1 468 932 no data 2 Aptostichus sp. AS217 C 2 469 933 3 3 Aptostichus sp. AS220 C 3 470 934 3 4 Araneus diadematus C 4 471 935 3 5 Araneus diadematus C 5 472 936 no data 6 Araneus diadematus C 6 473 937 no data 7 Araneus diadematus C 7 474 938 3 8 Atypoides riversi C 8 475 939 2 9 Bothriocyrtum californicum C 9 476 940 2 10 Bothriocyrtum californicum C 10 477 941 3 11 Bothriocyrtum californicum C 11 478 942 2 12 Deinopis Spinosa C 12 479 943 3 13 Deinopis Spinosa C 13 480 944 3 14 Deinopis Spinosa C 14 481 945 2 15 Dolomedes tenebrosus C 15 482 946 2 16 Euagrus chisoseus C 16 483 947 3 17 Plectreurys tristis C 17 484 948 3 18 Plectreurys tristis C 18 485 949 2 19 Plectreurys tristis C 19 486 950 1 20 Plectreurys tristis C 20 487 951 3 21 Agelenopsis aperta C 21 488 952 2 22 Araneus gemmoides C 22 489 953 3 23 Argiope argentata C 23 490 954 1 24 Argiope aurantia C 24 491 955 no data 25 Argiope bruennichi C 25 492 956 no data 26 Argiope bruennichi C 26 493 957 1 27 Atypoides riversi C 27 494 958 1 28 Avicularia juruensis C 28 495 959 1 29 Deinopis Spinosa C 29 496 960 2 30 Latrodectus hesperus C 30 497 961 2 31 Nephila antipodiana C 31 498 962 2 32 Nephila clavata C 32 499 963 2 33 Nephila clavipes C 33 500 964 1 34 Nephilengys cruentata C 34 501 965 3 35 Uloborus diversus C 35 502 966 no data 36 Araneus ventricosus C 36 503 967 3 37 Argiope argentata C 37 504 968 3 38 Deinopis spinosa C 38 505 969 2 39 Latrodectus hesperus C 39 506 970 3 40 Metepeira grandiosa C 40 507 971 3 41 Nephila antipodiana C 41 508 972 3 42 Nephila clavipes C 42 509 973 3 43 Nephilengys cruentata C 43 510 974 1 44 Parawixia bistriata C 44 511 975 3 45 Uloborus diversus C 45 512 976 2 46 Araneus ventricosus C 46 513 977 no data 47 Argiope trifasciata C 47 514 978 3 48 Nephila clavipes C 48 515 979 3 49 Nephilengys cruentata C 49 516 980 3 50 Nephila madagascariensis C 50 517 981 3 51 Latrodectus hesperus C 51 518 982 2 52 Araneus ventricosus C 52 519 983 2 53 Argiope trifasciata C 53 520 984 2 54 Parawixia bistriata C 54 521 985 3 55 Uloborus diversus C 55 522 986 1 56 Agelenopsis aperta C 56 523 987 3 57 Aphonopelma seemanni C 57 524 988 1 58 Araneus bicentenarius C 58 525 989 3 59 Araneus ventricosus C 59 526 990 2 60 Argiope amoena C 60 527 991 3 61 Argiope amoena C 61 528 992 no data 62 Argiope amoena C 62 529 993 3 63 Argiope amoena C 63 530 994 2 64 Argiope aurantia C 64 531 995 2 65 Argiope bruennichi C 65 532 996 2 66 Argiope bruennichi C 66 533 997 3 67 Argiope trifasciata C 67 534 998 3 68 Argiope trifasciata C 68 535 999 2 69 Avicularia juruensis C 69 536 1000 2 70 Avicularia juruensis C 70 537 1001 3 71 Avicularia juruensis C 71 538 1002 3 72 Deinopis spinosa C 72 539 1003 1 73 Deinopis spinosa C 73 540 1004 2 74 Deinopis spinosa C 74 541 1005 no data 75 Diguetia canities C 75 542 1006 2 76 Diguetia canities C 76 543 1007 3 77 Dolomedes tenebrosus C 77 544 1008 3 78 Euprosthenops australis C 78 545 1009 3 79 Euprosthenops australis C 79 546 1010 2 80 Euprosthenops australis C 80 547 1011 2 81 Gasteracantha mammosa C 81 548 1012 3 82 Hypochilus thorelli C 82 549 1013 2 83 Megahexura fulva C 83 550 1014 2 84 Nephila antipodiana C 84 551 1015 3 85 Nephila clavipes C 85 552 1016 3 86 Nephila clavipes C 86 553 1017 no data 87 Nephila madagascariensis C 87 554 1018 3 88 Nephila madagascariensis C 88 555 1019 3 89 Nephila pilipes C 89 556 1020 3 90 Nephila senegalensis C 90 557 1021 3 91 Nephilengys cruentata C 91 558 1022 2 92 Parawixia bistriata C 92 559 1023 3 93 Parawixia bistriata C 93 560 1024 2 94 Peucetia viridans C 94 561 1025 2 95 Poecilotheria regalis C 95 562 1026 1 96 Tetragnatha kauaiensis C 96 563 1027 1 97 Tetragnatha versicolor C 97 564 1028 2 98 Uloborus diversus C 98 565 1029 3 99 Araneus diadematus C 99 566 1030 1 100 Araneus diadematus C 100 567 1031 3 101 Araneus diadematus C 101 568 1032 2 102 Araneus diadematus C 102 569 1033 3 103 Araneus diadematus C 103 570 1034 3 104 Araneus diadematus C 104 571 1035 3 105 Araneus diadematus C 105 572 1036 2 106 Araneus diadematus C 106 573 1037 3 107 Araneus diadematus C 107 574 1038 3 108 Agelenopsis aperta N 108 575 1039 3 109 Argiope argentata N 109 576 1040 3 110 Argiope bruennichi N 110 577 1041 1 111 Argiope bruennichi N 111 578 1042 2 112 Latrodectus hesperus N 112 579 1043 1 113 Nephila clavata N 113 580 1044 3 114 Araneus ventricosus N 114 581 1045 3 115 Metepeira grandiosa N 115 582 1046 3 116 Uloborus diversus N 116 583 1047 3 117 Nephila clavipes N 117 584 1048 3 118 Nephila madagascariensis N 118 585 1049 3 119 Latrodectus hesperus N 119 586 1050 2 120 Latrodectus hesperus N 120 587 1051 2 121 Agelenopsis aperta N 121 588 1052 1 122 Argiope bruennichi N 122 589 1053 3 123 Argiope trifasciata N 123 590 1054 3 124 Bothriocyrtum californicum N 124 591 1055 2 125 Deinopis spinosa N 125 592 1056 3 126 Diguetia canities N 126 593 1057 3 127 Diguetia canities N 127 594 1058 3 128 Euprosthenops australis N 128 595 1059 3 129 Kukulcania hibernalis N 129 596 1060 1 130 Kukulcania hibernalis N 130 597 1061 3 131 Nephila clavipes N 131 598 1062 3 132 Nephila clavipes N 132 599 1063 3 133 Nephila clavipes N 133 600 1064 3 134 Nephila madagascariensis N 134 601 1065 3 135 Araneus diadematus N 135 602 1066 3 136 Araneus diadematus N 136 603 1067 2 137 Araneus diadematus N 137 604 1068 3 138 Araneus diadematus N 138 605 1069 2 139 Araneus diadematus N 139 606 1070 2 140 Araneus diadematus N 140 607 1071 3 141 Araneus diadematus N 141 608 1072 1 142 Araneus diadematus N 142 609 1073 3 143 Araneus diadematus N 143 610 1074 2 144 Araneus diadematus N 144 611 1075 2 145 Araneus diadematus N 145 612 1076 3 146 Aliatypus gulosus R 146 613 1077 3 147 Aliatypus gulosus R 147 614 1078 3 148 Aliatypus gulosus R 148 615 1079 3 149 Aliatypus gulosus R 149 616 1080 3 150 Aliatypus gulosus R 150 617 1081 3 151 Aliatypus gulosus R 151 618 1082 3 152 Aliatypus gulosus R 152 619 1083 3 153 Aptostichus sp. AS217 R 153 620 1084 3 154 Aptostichus sp. AS217 R 154 621 1085 3 155 Aptostichus sp. AS217 R 155 622 1086 3 156 Aptostichus sp. AS217 R 156 623 1087 3 157 Aptostichus sp. AS217 R 157 624 1088 3 158 Aptostichus sp. AS220 R 158 625 1089 2 159 Aptostichus sp. AS220 R 159 626 1090 3 160 Aptostichus sp. AS220 R 160 627 1091 3 161 Araneus diadematus R 161 628 1092 3 162 Araneus diadematus R 162 629 1093 2 163 Araneus diadematus R 163 630 1094 2 164 Araneus diadematus R 164 631 1095 2 165 Araneus diadematus R 165 632 1096 2 166 Atypoides riversi R 166 633 1097 3 167 Atypoides riversi R 167 634 1098 3 168 Atypoides riversi R 168 635 1099 2 169 Atypoides riversi R 169 636 1100 3 170 Atypoides riversi R 170 637 1101 no data 171 Atypoides riversi R 171 638 1102 1 172 Atypoides riversi R 172 639 1103 3 173 Bothriocyrtum californicum R 173 640 1104 3 174 Bothriocyrtum californicum R 174 641 1105 3 175 Bothriocyrtum californicum R 175 642 1106 3 176 Bothriocyrtum californicum R 176 643 1107 3 177 Bothriocyrtum californicum R 177 644 1108 3 178 Bothriocyrtum californicum R 178 645 1109 3 179 Bothriocyrtum californicum R 179 646 1110 3 180 Bothriocyrtum californicum R 180 647 1111 3 181 Bothriocyrtum californicum R 181 648 1112 3 182 Bothriocyrtum californicum R 182 649 1113 3 183 Deinopis Spinosa R 183 650 1114 3 184 Deinopis Spinosa R 184 651 1115 2 185 Deinopis Spinosa R 185 652 1116 3 186 Deinopis Spinosa R 186 653 1117 3 187 Deinopis Spinosa R 187 654 1118 3 188 Deinopis Spinosa R 188 655 1119 no data 189 Deinopis Spinosa R 189 656 1120 2 190 Deinopis Spinosa R 190 657 1121 3 191 Dolomedes tenebrosus R 191 658 1122 2 192 Dolomedes tenebrosus R 192 659 1123 no data 193 Dolomedes tenebrosus R 193 660 1124 3 194 Euagrus chisoseus R 194 661 1125 2 195 Euagrus chisoseus R 195 662 1126 2 196 Euagrus chisoseus R 196 663 1127 2 197 Plectreurys tristis R 197 664 1128 3 198 Plectreurys tristis R 198 665 1129 3 199 Plectreurys tristis R 199 666 1130 3 200 Plectreurys tristis R 200 667 1131 2 201 Plectreurys tristis R 201 668 1132 3 202 Plectreurys tristis R 202 669 1133 3 203 Plectreurys tristis R 203 670 1134 2 204 Plectreurys tristis R 204 671 1135 3 205 Plectreurys tristis R 205 672 1136 3 206 Plectreurys tristis R 206 673 1137 3 207 Plectreurys tristis R 207 674 1138 3 208 Plectreurys tristis R 208 675 1139 2 209 Plectreurys tristis R 209 676 1140 3 210 Plectreurys tristis R 210 677 1141 3 211 Plectreurys tristis R 211 678 1142 3 212 Plectreurys tristis R 212 679 1143 3 213 Plectreurys tristis R 213 680 1144 3 214 Plectreurys tristis R 214 681 1145 3 215 Plectreurys tristis R 215 682 1146 3 216 Agelenopsis aperta R 216 683 1147 3 217 Agelenopsis aperta R 217 684 1148 3 218 Araneus gemmoides R 218 685 1149 2 219 Araneus gemmoides R 219 686 1150 3 220 Araneus gemmoides R 220 687 1151 2 221 Argiope amoena R 221 688 1152 no data 222 Argiope amoena R 222 689 1153 3 223 Argiope argentata R 223 690 1154 2 224 Argiope argentata R 224 691 1155 2 225 Argiope argentata R 225 692 1156 2 226 Argiope aurantia R 226 693 1157 2 227 Argiope aurantia R 227 694 1158 2 228 Argiope aurantia R 228 695 1159 2 229 Argiope aurantia R 229 696 1160 2 230 Argiope bruennichi R 230 697 1161 2 231 Argiope bruennichi R 231 698 1162 2 232 Argiope bruennichi R 232 699 1163 2 233 Argiope bruennichi R 233 700 1164 2 234 Argiope bruennichi R 234 701 1165 3 235 Argiope bruennichi R 235 702 1166 2 236 Argiope bruennichi R 236 703 1167 2 237 Argiope bruennichi R 237 704 1168 2 238 Argiope bruennichi R 238 705 1169 2 239 Argiope bruennichi R 239 706 1170 3 240 Argiope bruennichi R 240 707 1171 2 241 Argiope bruennichi R 241 708 1172 2 242 Argiope bruennichi R 242 709 1173 3 243 Argiope bruennichi R 243 710 1174 2 244 Argiope bruennichi R 244 711 1175 3 245 Argiope bruennichi R 245 712 1176 2 246 Argiope bruennichi R 246 713 1177 2 247 Argiope bruennichi R 247 714 1178 3 248 Argiope bruennichi R 248 715 1179 2 249 Argiope bruennichi R 249 716 1180 2 250 Atypoides riversi R 250 717 1181 2 251 Atypoides riversi R 251 718 1182 2 252 Atypoides riversi R 252 719 1183 3 253 Atypoides riversi R 253 720 1184 1 254 Atypoides riversi R 254 721 1185 2 255 Atypoides riversi R 255 722 1186 2 256 Atypoides riversi R 256 723 1187 2 257 Avicularia juruensis R 257 724 1188 2 258 Avicularia juruensis R 258 725 1189 1 259 Avicularia juruensis R 259 726 1190 1 260 Deinopis Spinosa R 260 727 1191 3 261 Deinopis Spinosa R 261 728 1192 3 262 Deinopis Spinosa R 262 729 1193 2 263 Latrodectus hesperus R 263 730 1194 3 264 Latrodectus hesperus R 264 731 1195 3 265 Latrodectus hesperus R 265 732 1196 2 266 Latrodectus hesperus R 266 733 1197 1 267 Latrodectus hesperus R 267 734 1198 1 268 Latrodectus hesperus R 268 735 1199 2 269 Nephila antipodiana R 269 736 1200 3 270 Nephila clavata R 270 737 1201 2 271 Nephila clavata R 271 738 1202 no data 272 Nephila clavata R 272 739 1203 2 273 Nephila clavata R 273 740 1204 2 274 Nephila clavata R 274 741 1205 1 275 Nephila clavata R 275 742 1206 1 276 Nephila clavata R 276 743 1207 2 277 Nephila clavata R 277 744 1208 1 278 Nephila clavipes R 278 745 1209 2 279 Nephila clavipes R 279 746 1210 2 280 Nephilengys cruentata R 280 747 1211 no data 281 Uloborus diversus R 281 748 1212 3 282 Uloborus diversus R 282 749 1213 1 283 Uloborus diversus R 283 750 1214 3 284 Uloborus diversus R 284 751 1215 1 285 Araneus ventricosus R 285 752 1216 2 286 Araneus ventricosus R 286 753 1217 3 287 Araneus ventricosus R 287 754 1218 2 288 Araneus ventricosus R 288 755 1219 2 289 Araneus ventricosus R 289 756 1220 3 290 Araneus ventricosus R 290 757 1221 2 291 Araneus ventricosus R 291 758 1222 3 292 Araneus ventricosus R 292 759 1223 3 293 Argiope argentata R 293 760 1224 3 294 Deinopis spinosa R 294 761 1225 2 295 Latrodectus hesperus R 295 762 1226 3 296 Latrodectus hesperus R 296 763 1227 3 297 Metepeira grandiosa R 297 764 1228 2 298 Metepeira grandiosa R 298 765 1229 3 299 Nephila antipodiana R 299 766 1230 2 300 Nephila clavipes R 300 767 1231 3 301 Nephila clavipes R 301 768 1232 3 302 Nephila clavipes R 302 769 1233 2 303 Nephila clavipes R 303 770 1234 3 304 Nephilengys cruentata R 304 771 1235 2 305 Nephilengys cruentata R 305 772 1236 3 306 Nephilengys cruentata R 306 773 1237 3 307 Nephilengys cruentata R 307 774 1238 no data 308 Nephilengys cruentata R 308 775 1239 3 309 Nephilengys cruentata R 309 776 1240 2 310 Nephilengys cruentata R 310 777 1241 3 311 Nephilengys cruentata R 311 778 1242 3 312 Nephilengys cruentata R 312 779 1243 2 313 Parawixia bistriata R 313 780 1244 3 314 Parawixia bistriata R 314 781 1245 3 315 Uloborus diversus R 315 782 1246 3 316 Uloborus diversus R 316 783 1247 3 317 Uloborus diversus R 317 784 1248 3 318 Uloborus diversus R 318 785 1249 2 319 Araneus ventricosus R 319 786 1250 2 320 Argiope trifasciata R 320 787 1251 3 321 Argiope trifasciata R 321 788 1252 3 322 Argiope trifasciata R 322 789 1253 3 323 Nephila clavipes R 323 790 1254 2 324 Nephila clavipes R 324 791 1255 3 325 Nephila clavipes R 325 792 1256 3 326 Nephila clavipes R 326 793 1257 3 327 Nephila clavipes R 327 794 1258 3 328 Nephila clavipes R 328 795 1259 3 329 Nephilengys cruentata R 329 796 1260 3 330 Nephilengys cruentata R 330 797 1261 2 331 Nephilengys cruentata R 331 798 1262 1 332 Nephila madagascariensis R 332 799 1263 2 333 Nephila madagascariensis R 333 800 1264 3 334 Nephila madagascariensis R 334 801 1265 2 335 Nephila madagascariensis R 335 802 1266 3 336 Nephila madagascariensis R 336 803 1267 1 337 Nephila madagascariensis R 337 804 1268 no data 338 Nephila madagascariensis R 338 805 1269 2 339 Nephila madagascariensis R 339 806 1270 2 340 Latrodectus hesperus R 340 807 1271 3 341 Latrodectus hesperus R 341 808 1272 2 342 Latrodectus hesperus R 342 809 1273 3 343 Latrodectus hesperus R 343 810 1274 2 344 Latrodectus hesperus R 344 811 1275 no data 345 Latrodectus hesperus R 345 812 1276 2 346 Latrodectus hesperus R 346 813 1277 3 347 Latrodectus hesperus R 347 814 1278 3 348 Latrodectus hesperus R 348 815 1279 3 349 Latrodectus hesperus R 349 816 1280 2 350 Argiope amoena R 350 817 1281 3 351 Argiope amoena R 351 818 1282 3 352 Argiope amoena R 352 819 1283 3 353 Argiope amoena R 353 820 1284 3 354 Araneus ventricosus R 354 821 1285 3 355 Araneus ventricosus R 355 822 1286 3 356 Araneus ventricosus R 356 823 1287 3 357 Araneus ventricosus R 357 824 1288 3 358 Araneus ventricosus R 358 825 1289 3 359 Araneus ventricosus R 359 826 1290 3 360 Araneus ventricosus R 360 827 1291 3 361 Araneus ventricosus R 361 828 1292 3 362 Argiope trifasciata R 362 829 1293 3 363 Argiope trifasciata R 363 830 1294 3 364 Argiope trifasciata R 364 831 1295 3 365 Argiope trifasciata R 365 832 1296 3 366 Argiope trifasciata R 366 833 1297 3 367 Argiope trifasciata R 367 834 1298 3 368 Argiope trifasciata R 368 835 1299 3 369 Argiope trifasciata R 369 836 1300 3 370 Parawixia bistriata R 370 837 1301 3 371 Parawixia bistriata R 371 838 1302 3 372 Uloborus diversus R 372 839 1303 3 373 Uloborus diversus R 373 840 1304 3 374 Uloborus diversus R 374 841 1305 3 375 Uloborus diversus R 375 842 1306 3 376 Agelenopsis aperta R 376 843 1307 3 377 Agelenopsis aperta R 377 844 1308 3 378 Agelenopsis aperta R 378 845 1309 2 379 Agelenopsis aperta R 379 846 1310 2 380 Aphonopelma seemanni R 380 847 1311 3 381 Araneus ventricosus R 381 848 1312 3 382 Argiope aurantia R 382 849 1313 3 383 Argiope bruennichi R 383 850 1314 3 384 Argiope bruennichi R 384 851 1315 3 385 Argiope bruennichi R 385 852 1316 3 386 Argiope bruennichi R 386 853 1317 3 387 Argiope bruennichi R 387 854 1318 3 388 Argiope bruennichi R 388 855 1319 3 389 Argiope bruennichi R 389 856 1320 3 390 Argiope bruennichi R 390 857 1321 3 391 Argiope bruennichi R 391 858 1322 3 392 Argiope bruennichi R 392 859 1323 3 393 Argiope bruennichi R 393 860 1324 3 394 Argiope trifasciata R 394 861 1325 3 395 Argiope trifasciata R 395 862 1326 3 396 Argiope trifasciata R 396 863 1327 1 397 Argiope trifasciata R 397 864 1328 2 398 Argiope trifasciata R 398 865 1329 1 399 Argiope trifasciata R 399 866 1330 3 400 Argiope trifasciata R 400 867 1331 1 401 Avicularia juruensis R 401 868 1332 3 402 Avicularia juruensis R 402 869 1333 no data 403 Avicularia juruensis R 403 870 1334 3 404 Deinopis spinosa R 404 871 1335 3 405 Deinopis spinosa R 405 872 1336 2 406 Deinopis spinosa R 406 873 1337 3 407 Deinopis spinosa R 407 874 1338 2 408 Deinopis spinosa R 408 875 1339 no data 409 Deinopis spinosa R 409 876 1340 3 410 Diguetia canities R 410 877 1341 3 411 Diguetia canities R 411 878 1342 3 412 Diguetia canities R 412 879 1343 3 413 Dolomedes tenebrosus R 413 880 1344 2 414 Dolomedes tenebrosus R 414 881 1345 3 415 Dolomedes tenebrosus R 415 882 1346 3 416 Euprosthenops australis R 416 883 1347 2 417 Euprosthenops australis R 417 884 1348 1 418 Euprosthenops australis R 418 885 1349 3 419 Euprosthenops australis R 419 886 1350 2 420 Euprosthenops australis R 420 887 1351 3 421 Euprosthenops australis R 421 888 1352 3 422 Euprosthenops australis R 422 889 1353 3 423 Euprosthenops australis R 423 890 1354 3 424 Euprosthenops australis R 424 891 1355 3 425 Gasteracantha mammosa R 425 892 1356 1 426 Hypochilus thorelli R 426 893 1357 3 427 Hypochilus thorelli R 427 894 1358 3 428 Kukulcania hibernalis R 428 895 1359 3 429 Kukulcania hibernalis R 429 896 1360 3 430 Megahexura fulva R 430 897 1361 no data 431 Megahexura fulva R 431 898 1362 3 432 Megahexura fulva R 432 899 1363 no data 433 Megahexura fulva R 433 900 1364 3 434 Megahexura fulva R 434 901 1365 3 435 Megahexura fulva R 435 902 1366 3 436 Nephila clavipes R 436 903 1367 1 437 Nephila clavipes R 437 904 1368 3 438 Nephila clavipes R 438 905 1369 3 439 Nephila clavipes R 439 906 1370 3 440 Nephila clavipes R 440 907 1371 1 441 Nephila madagascariensis R 441 908 1372 3 442 Nephila madagascariensis R 442 909 1373 3 443 Nephila madagascariensis R 443 910 1374 3 444 Nephila madagascariensis R 444 911 1375 3 445 Nephila madagascariensis R 445 912 1376 2 446 Nephila madagascariensis R 446 913 1377 2 447 Nephila madagascariensis R 447 914 1378 2 448 Nephila madagascariensis R 448 915 1379 2 449 Nephila madagascariensis R 449 916 1380 2 450 Nephila pilipes R 450 917 1381 no data 451 Nephilengys cruentata R 451 918 1382 3 452 Nephilengys cruentata R 452 919 1383 2 453 Parawixia bistriata R 453 920 1384 2 454 Parawixia bistriata R 454 921 1385 2 455 Parawixia bistriata R 455 922 1386 3 456 Parawixia bistriata R 456 923 1387 2 457 Peucetia viridans R 457 924 1388 3 458 Poecilotheria regalis R 458 925 1389 2 459 Poecilotheria regalis R 459 926 1390 2 460 Poecilotheria regalis R 460 927 1391 no data 461 Tetragnatha kauaiensis R 461 928 1392 2 462 Uloborus diversus R 462 929 1393 1 RM409 Argiope bruennichi R 463 930 1394 no data RM410 Argiope bruennichi R 464 931 1395 no data RM411 Argiope bruennichi R 465 N/A 1396 no data RM434 Argiope bruennichi R 466 N/A 1397 no data RM439 Argiope bruennichi R 467 N/A 1398 3

Example 4 Amplification of N, R, and C Sequences for Insertion into an Assembly Vector

The DNA for N, R, and C sequences were PCR amplified from the expression vector and ligated into assembly vectors using AscI/SbfI restriction sites.

The forward primer consisted of the sequence: 5′-CTAAGAGGCGCGCCTAAGCGATGGTCTCAA-3′ (SEQ ID NO: 2827)+the first 19 bp of the N, R, or C sequence.

The reverse primer consisted of the last 17 bp of the N, R, or C sequence+3′-GGTACGTCTTCATCGCTATCCTGCAGGCTACGT-5′ (SEQ ID NO: 2828).

For example, for sequence:

(SEQ ID NO: 4) GGTGCAGGTGCAAGGGCTGCTGGAGGCTACGGTGGAGGATACGGTGCCGG TGCGGGTGCAGGAGCCGGCGCCGCAGCTTCCGCCGGAGCCTCCGGTGGAT ACGGAGGTGGATATGGTGGCGGAGCTGGTGCTGGTGCCGTAGCAGGTGCC TCAGCTGGAAGCTACGGAGGTGCTGTTAATAGACTGAGTTCCGCAGGTGC AGCCTCTAGAGTGTCGTCCAACGTCGCAGCCATTGCATCTGCTGGTGCTG CCGCTTTGCCCAACGTTATTTCCAACATCTATAGTGGTGTTCTTTCATCT GGCGTGTCATCCTCCGAAGCACTTATTCAGGCTTTGTTAGAAGTAATCAG TGCTTTAATTCATGTCTTAGGATCAGCTTCTATCGGCAACGTTTCATCTG TTGGTGTTAATTCCGCACTTAATGCTGTGCAAAACGCCGTAGGCGCCTAT GCCGGA  the primers used were:

Fwd:  (SEQ ID NO: 2829) 5′-CTAAGAGGCGCGCCTAAGCGATGGTCTCAAGGTGCAGGTGCAAGGG  CTG-3′ Rev:  (SEQ ID NO: 2830) 3′-TAGGCGCCTATGCCGGAGGTACGTCTTCATCGCTATCCTGCAGGCTA CGT-5′

The PCR reaction solution consisted of 12.5 μL 2×KOD Extreme Buffer, 0.25 μl KOD Extreme Hot Start Polymerase, 0.5 μl 10 μM Fwd oligo, 0.5 μl 10 μM Rev oligo, 5 ng template DNA (expression vector), 0.5 μl of 10 mM dNTPs, and ddH2O added to final volume of 25 The reaction was then thermocycled according to the program:

-   -   1. Denature at 94° C. for 5 minutes     -   2. Denature at 94° C. for 30 seconds     -   3. Anneal at 55° C. for 30 seconds     -   4. Extend at 72° C. for 30 seconds     -   5. Repeat steps 2-4 for 29 additional cycles     -   6. Final extension at 72° C. for 5 minutes         Resulting PCR products were digested with restriction enzymes         AscI and SbfI, and ligated into an assembly vector (see         description in Example 5), one of KC (RM396, SEQ ID NO:1402), KA         (RM397, SEQ ID NO:1403), AC (RM398, SEQ ID NO:1404), AK (RM399,         SEQ ID NO:1405), CA (RM400, SEQ ID NO:1406), or CK (RM401, SEQ         ID NO:1407) that had been digested with the same enzymes to         release an unwanted insert using routine methods.

Example 5 Synthesis of Silk from Argiope Bruennichi MaSp2 Blocks (RM439, “18B”)

Using the algorithm described in Example 2, a set of 6 repeat blocks (or block co-polymer) from Argiope bruennichi MaSp2 were selected and divided into 2 R sequences consisting of 3 blocks each. The two 3-block R sequences were then synthesized from short oligonucleotides as follows:

Synthesis of RM409 Sequence:

The Argiope bruennichi MaSp2 block sequences were generated using methodology distinct from that employed in Example 3. Oligos RM2919-RM2942 (SEQ ID NOs: 1468-1491) in Table 3 were combined into a single mixture with equal amounts of each oligo, 100 μM in total. The oligos were phosphorylated in a phosphorylation reaction prepared by combining 1 μl 10×NEB T4 DNA ligase buffer, 1 μl 100 μM pooled oligos, 1 μl NEB T4 Polynucleotide Kinase (10,000 U/ml), and 7 μl ddH2O and incubating for 1 hour at 37° C. The oligos were then annealed by mixing 4 μl of the phosphorylation reaction with 16 μl of ddH2O, heating the mixture to 95° C. for 5 minutes, and then cooling the mixture to 25° C. at a rate of 0.1° C./sec. The oligos were then ligated together into a vector by combining 4 μl of the annealed oligos with 5 nmol vector backbone (RM396 [SEQ ID NO: 1405], digested with AscI and SbfI), 1 μl NEB T4 DNA ligase (400,000 U/ml), 1 μl 10×NEB T4 DNA ligase buffer, and ddH2O to 10 The ligation solution was incubated for 30 minutes at room temperature. The entirety of the ligation reaction was transformed into E. coli for clonal selection, plasmid isolation, and sequence verification according to known techniques.

The resulting oligonucleotide has a 5′ to 3′ nucleotide sequence of SEQ ID NO: 930 and is identified as RM409.

TABLE 3 Oligo sequences for generating RM409 silk repeat domain (with flanking sequences for cloning) (SEQ ID NO: 930) SEQ ID NO:  ID 5′ to 3′ Nucleotide Sequence 1469 RM2919 CGCGCCTTAGCGATGGTCTCAAGGTGGTTACGGTCCAGGCGCTGGTCAACAAGGTCCA 1470 RM2920 GGAAGTGGTGGTCAACAAGGACCTGGCGGTCAAGGACCCTACGGTAGTGG 1471 RM2921 CCAACAAGGTCCAGGTGGAGCAGGACAGCAGGGTCCGGGAGGCCAAGGAC 1472 RM2922 CTTACGGACCAGGTGCTGCTGCTGCCGCCGCTGCCGCTGCCGGAGGTTACGGT 1473 RM2923 CCAGGAGCCGGACAACAGGGTCCAGGTGGAGCTGGACAACAAGGTCC 1474 RM2924 AGGATCACAAGGTCCTGGTGGACAAGGTCCATACGGTCCTGGTGCTGGTC 1475 RM2925 AACAGGGACCAGGTAGTCAAGGACCTGGTTCAGGTGGTCAGCAGGGTCCAG 1476 RM2926 GAGGACAGGGTCCTTACGGCCCTTCTGCCGCTGCAGCAGCAGCCGCTG 1477 RM2927 CCGCAGGAGGATACGGACCTGGTGCTGGACAACGATCTCAAGGACCAGG 1478 RM2928 AGGACAAGGTCCTTATGGACCTGGCGCTGGCCAACAAGGACCTGGTTCT 1479 RM2929 CAGGGTCCAGGTTCAGGAGGCCAACAAGGCCCAGGAGGTCAAGGACCAT 1480 RM2930 ACGGACCATCCGCTGCGGCAGCTGCAGCTGCTGCAGGTACGTCTTCATCGCTATCCTGCA 1481 RM2931 ACTTCCTGGACCTTGTTGACCAGCGCCTGGACCGTAACCACCTTGAGACCATCGCTAAGG 1482 RM2932 TGTTGGCCACTACCGTAGGGTCCTTGACCGCCAGGTCCTTGTTGACCACC 1483 RM2933 CGTAAGGTCCTTGGCCTCCCGGACCCTGCTGTCCTGCTCCACCTGGACCT 1484 RM2934 TCCTGGACCGTAACCTCCGGCAGCGGCAGCGGCGGCAGCAGCAGCACCTGGTC 1485 RM2935 GATCCTGGACCTTGTTGTCCAGCTCCACCTGGACCCTGTTGTCCGGC 1486 RM2936 CCTGTTGACCAGCACCAGGACCGTATGGACCTTGTCCACCAGGACCTTGT 1487 RM2937 GTCCTCCTGGACCCTGCTGACCACCTGAACCAGGTCCTTGACTACCTGGTC 1488 RM2938 CTGCGGCAGCGGCTGCTGCTGCAGCGGCAGAAGGGCCGTAAGGACCCT 1489 RM2939 TGTCCTCCTGGTCCTTGAGATCGTTGTCCAGCACCAGGTCCGTATCCTC 1490 RM2940 ACCCTGAGAACCAGGTCCTTGTTGGCCAGCGCCAGGTCCATAAGGACCT 1491 RM2941 GTCCGTATGGTCCTTGACCTCCTGGGCCTTGTTGGCCTCCTGAACCTGG 1492 RM2942 GGATAGCGATGAAGACGTACCTGCAGCAGCTGCAGCTGCCGCAGCGGATG

Synthesis of RM410 Sequence:

Oligos RM2999-RM3014 (SEQ ID NOs: 1492-1507) in Table 4 were combined into a single mixture at a concentration of 100 μM of each oligo. The oligos were phosphorylated in a phosphorylation reaction prepared by combining 1 μl 10×NEB T4 DNA ligase buffer, 1 μl 100 μM pooled oligos, 1 μl NEB T4 Polynucleotide Kinase (10,000 U/ml), and 7 μl ddH2O and incubating for 1 hour at 37° C. The oligos were then annealed by mixing 4 μl of the phosphorylation reaction with 16 μl of ddH2O, heating the mixture to 95° C. for 5 minutes, and then cooling the mixture to 25° C. at a rate of 0.1° C./sec. The oligos were then ligated together into a vector by combining 4 μl of the annealed oligos with 5 nmol vector backbone (RM400 [SEQ ID NO: 1406], digested with AscI and SbfI), 1 μl NEB T4 DNA ligase (400,000 U/ml), 1 μl 10×NEB T4 DNA ligase buffer, and ddH2O to 10 The ligation solution was incubated for 30 minutes at room temperature. The entirety of the ligation reaction was transformed into E. coli for clonal selection, plasmid isolation, and sequence verification according to known techniques.

The resulting oligonucleotide has a 5′ to 3′ nucleotide sequence of SEQ ID NO: 931 and is identified as RM410.

TABLE 4 Oligo sequences for generating RM410 silk repeat domain (with flanking sequences for cloning) (SEQ ID NO: 931) SEQ ID NO: ID 5′ to 3′ Nucleotide Sequence 1493 RM2999 CGCGCCTTAGCGATGGTCTCAAGGTGGATATGGCCCAGGAGCCGGACAACAGGGTCCT 1494 RM3000 GGTTCACAAGGTCCAGGATCTGGTGGTCAACAGGGACCAGGCGGCCAGGGAC 1495 RM3001 CTTATGGTCCAGGAGCCGCTGCAGCAGCAGCAGCTGTTGGAGGTTACGGCC 1496 RM3002 CTGGTGCCGGTCAACAAGGCCCAGGATCTCAGGGTCCTGGATCTGGAGGAC 1497 RM3003 AACAAGGTCCTGGAGGTCAGGGTCCATACGGACCTTCAGCAGCAGCTGCTGC 1498 RM3004 TGCAGCCGCTGGTGGTTATGGACCTGGTGCTGGTCAACAAGGACCGGGTT 1499 RM3005 CTCAGGGTCCGGGTTCAGGAGGTCAGCAGGGCCCTGGTGGACAAGGACCTT 1500 RM3006 ATGGACCTAGTGCGGCTGCAGCAGCTGCCGCCGCAGGTACGTCTTCATCGCTATCCTGCA 1501 RM3007 TGAACCAGGACCCTGTTGTCCGGCTCCTGGGCCATATCCACCTTGAGACCATCGCTAAGG 1502 RM3008 CATAAGGTCCCTGGCCGCCTGGTCCCTGTTGACCACCAGATCCTGGACCTTG 1503 RM3009 CACCAGGGCCGTAACCTCCAACAGCTGCTGCTGCTGCAGCGGCTCCTGGAC 1504 RM3010 CTTGTTGTCCTCCAGATCCAGGACCCTGAGATCCTGGGCCTTGTTGACCGG 1505 RM3011 GCTGCAGCAGCAGCTGCTGCTGAAGGTCCGTATGGACCCTGACCTCCAGGAC 1506 RM3012 CCTGAGAACCCGGTCCTTGTTGACCAGCACCAGGTCCATAACCACCAGCG 1507 RM3013 GTCCATAAGGTCCTTGTCCACCAGGGCCCTGCTGACCTCCTGAACCCGGAC 1508 RM3014 GGATAGCGATGAAGACGTACCTGCGGCGGCAGCTGCTGCAGCCGCACTAG

Assembly and Assay of Argiope bruennichi MaSp2, “18B”

RM409 (SEQ ID NO: 930) and RM410 (SEQ ID NO: 931) oligonucleotide sequences synthesized according to the method described above were assembled according to the diagram shown in FIG. 6 to generate RM439 silk nucleotide sequence (e.g., “18B”).

RM409 (SEQ ID NO: 930) and RM410 (SEQ ID NO: 931) in assembly vectors were digested and ligated according to the diagrams shown in FIG. 7 and FIG. 8. Silk N, R, and C domains, as well as additional elements including the alpha mating factor pre-pro sequence and a 3× FLAG tag, were assembled using a pseudo-scarless 2 antibiotic (2ab) method (Leguia, M., et al., 2ab assembly: a methodology for automatable, high-throughput assembly of standard biological parts, J. Biol. Eng., 7:1 (2013); and Kodumal, S. J., et al., Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster, Proc. Natl. Acad. Sci. U.S.A., 101:44, pg. 15573-15578 (2004)).

2ab assembly relies on the use of 6 assembly vectors that are identical except for the identity and relative position of 2 selectable markers. Each vector is resistant to exactly 2 of: chloramphenicol (CamR), kanamycin (KanR), and ampicillin (AmpR). The order (relative position) of the resistance genes matters, such that AmpR/KanR is distinct from KanR/AmpR for the purpose of DNA assembly. The 6 assembly vectors are shown in Table 5, are named based on the two resistance markers in each (C for CamR, K for KanR, and A for AmpR). The 6 assembly vectors are as follows: KC (RM396, SEQ ID NO:1402), KA (RM397, SEQ ID NO:1403), AC (RM398, SEQ ID NO:1404), AK (RM399, SEQ ID NO:1405), CA (RM400, SEQ ID NO:1406), and CK (RM401, SEQ ID NO:1407). Assembly vectors are shown in Table 5. Sequences for the vectors include those of SEQ ID NOs: 1399-1410.

TABLE 5 Expression and assembly vectors Vector ID Vector Type Description SEQ ID NO: RM618 Expression Vector circular, double 1399 (dummy insert) stranded DNA RM652 Expression Vector circular, double 1400 (dummy insert) stranded DNA RM468 Expression Vector circular, double 1401 (dummy insert) stranded DNA RM396 Assembly Vector circular, double 1402 (dummy insert) stranded DNA RM397 Assembly Vector circular, double 1403 (dummy insert) stranded DNA RM398 Assembly Vector circular, double 1404 (dummy insert) stranded DNA RM399 Assembly Vector circular, double 1405 (dummy insert) stranded DNA RM400 Assembly Vector circular, double 1406 (dummy insert) stranded DNA RM401 Assembly Vector circular, double 1407 (dummy insert) stranded DNA RM529 Assembly Vector, alpha circular, double 1408 mating factor special case stranded DNA

FIG. 7 shows a single assembly reaction performed with two compatible vectors, AC (RM398 SEQ ID NO:1404) and CK (RM401 SEQ ID NO:1407), one containing a sequence destined for the 5′ end of the target composite sequence and one destined for the 3′ end of the target composite sequence. The plasmid bearing the 5′ sequence is independently digested with BbsI, while the plasmid bearing the 3′ sequence is independently digested with BsaI.

After inactivation of the enzymes, the two digested plasmids are pooled and ligated. The desired product resides in an AK vector, which is distinct from all input vectors and undesired byproducts. This enables selection for the desired product after transformation into E. coli.

The DNA sequence of the cloning sites during this process is shown in FIG. 8. By selecting the 4 bp overhang generated by the type IIs enzymes to be AGGT, assembly of DNA fragments generates scarless junctions in the desired encoded polypeptide provided that the polypeptide starts with a glycine (coded by GGT) and terminates with a codon ending in an A (all except F, Y, W, C, H, N, M, and D).

The assembly of RM409 (SEQ ID NO: 930) and RM410 (SEQ ID NO: 931) in KC and CA assembly vectors, respectively, generated RM411(SEQ ID NO: 465) in KA, as shown in FIG. 6. The RM411(SEQ ID NO: 465) sequence was transferred to AC and CA using AscI and SbfI. The RM411(SEQ ID NO: 465) KA and AC sequences were digested and ligated according to the procedure described above to generate RM434 (SEQ ID NO: 466) in KC. Finally, RM434 (SEQ ID NO: 466) in KC was digested and ligated with RM411 (SEQ ID NO: 465) in CA to generate the final silk polypeptide coding sequence, RM439 (SEQ ID NO: 467) (aka, “18B”).

Transfer of “18B” Silk Polypeptide Coding Sequence (RM439) to the RM468 Expression Vector:

The RM468 (SEQ ID NO: 1401) expression vector contains an alpha mating factor sequence and a 3× FLAG sequence (SEQ ID NO: 1409). The 18B silk polypeptide coding sequence RM439 (SEQ ID NO: 467) was transferred to the RM468 (SEQ ID NO: 1401) expression vector via BtgZI restriction enzymes and Gibson reaction kits. The RM439 vector was digested with BtgZI, and the polynucleotide fragment containing the silk sequence isolated by gel electrophoresis. The expression vector, RM468, exclusive of an unwanted dummy insert, was amplified by PCR using primers RM3329 and RM3330, using the conditions described in Example 4. The resulting PCR product and isolated silk fragment were combined using a Gibson reaction kit according to the manufacturers instructions. Gibson reaction kits are commercially available (https://www.neb.com/products/e2611-gibson-assembly-master-mix), and are described in a U.S. Pat. No. 5,436,149 and in Gibson, D. G. et al., Enzymatic assembly of DNA molecules up to several hundred kilobases, Nat. Methods, 6:5, pg. 343-345 (2009).

The resulting expression vector containing RM439 (SEQ ID NO: 467) was transformed into Pichia (Komagataella) pastoris. Clones of the resulting cells were cultured according to the following conditions: The culture was grown in a minimal basal salt media, similar to one described in [http://tools.invitrogen.com/content/sfs/manuals/pichiaferm_prot.pdf] with 50 g/L of glycerol as a starting feedstock. Growth was in a stirred fermentation vessel controlled at 30 C, with 1 VVM of air flow and 2000 rpm agitation. pH was controlled at 3 with the on-demand addition of ammonium hydroxide. Additional glycerol was added as needed based on sudden increases in dissolved oxygen. Growth was allowed to continue until dissolved oxygen reached 15% of maximum at which time the culture was harvested, typically at 200-300 OD of cell density.

The broth from the fermenter was decellularized by centrifugation. The supernatant from the Pichia (Komagataella) pastoris culture was collected. Low molecular weight components were removed from the supernatant using ultrafiltration to remove particles smaller than the block copolymer polypeptides. The filtered culture supernatant was then concentrated up to 50×. The polypeptides in the supernatant were precipitated and analyzed via a western blot. The product is shown in the western blot in FIG. 9. The predicted molecular weight of processed 18B is 82 kDa. The product observed in the western blot in FIG. 9 exhibited a higher MW of ˜120 kDa. While the source of this discrepancy is unknown, other silk polypeptides have been observed to appear at a higher than expected molecular weight.

The 18B block copolymer polypeptide was purified and processed into a fiber spinnable solution. The fiber spinnable solution was prepared by dissolving the purified and dried polypeptide in a spinning solvent. The polypeptide is dissolved in the selected solvent at 20 to 30% by weight. The fiber spinnable solution was then extruded through a 150 micron diameter orifice into a coagulation bath comprising 90% methanol/10% water by volume. Fibers were removed from the coagulation and drawn from 1 to 5 times their length, and subsequently allowed to dry. The resulting fiber is shown in FIG. 10.

Mechanical testing was performed on the 18B block copolymer polypeptide that was secreted, purified, dissolved, and turned into a fiber as described above. Fibers were tested for mechanical properties on a custom-built tensile tester, using common processes. Test samples were mounted with a gauge length of 5.75 mm and tested at a strain rate of 1%. The resultant forces were normalized to the fiber diameter, as measured by microscopy. Results of stress vs strain are shown in FIG. 11 in which each stress-strain curve represents a replicate measurement from a fiber from a single spinning experiment, from a single batch.

Example 6 Assembly and Assay of 4× Repeat R Sequences

Selected R domains from SEQ ID NOs: 1-1398 that expressed and secreted well were concatenated into 4× repeat domains using the assembly scheme shown in FIG. 12. The concatenation was performed as described in Example 4 and shown in FIGS. 7 and 8. Selected sequences from this ligation of R sequences are shown in Table 6. Sequences for these silk constructs include those full-length silk construct sequences of SEQ ID NOs: 1411-1468. The resulting products comprising 4 repeat sequences, an alpha mating factor, and a 3× FLAG domain were digested with AscI and SbfI to release the desired silk sequence and ligated into expression vector RM652 (SEQ ID NO: 1400) that had been digested with AscI and SbfI to release an unwanted dummy insert. After clonal isolation from E. coli, vectors were then transformed into Pichia pastoris. Transformants were plated on YPD agar plates containing 25 μg/ml nourseothricin and incubated for 48 hours at 30° C. Three clones from each transformation were inoculated into 400 μl of BMGY in a 96-well square-well block, and incubated for 48 hours at 30° C. with agitation at 1000 rpm. Cells were pelleted via centrifugation, and the supernatant was recovered for analysis of block copolymer polypeptide content via western blot (FIG. 13). Of the 28 constructs transformed with 4× identical repeat sequences, most (18/28) had at least one clone with a substantial signal on the western blot, and only 1 showed no signal at all. Of two constructs composed of 2 repeats each of 2 distinct repeat sequences, one showed a strong western blot signal, while the other showed a modest western signal. This confirms that assembling larger block copolymer-expressing polynucleotides from smaller, well-expressed polynucleotides generally leads to functionally expressed block copolymer polypeptides. Streakiness, multiple bands, and clone-to-clone variation are evident on the western. While the specific source of these variations has not been identified, they are generally consistent with typically observed phenomena, including polypeptide degradation, post-translational modification (e.g., glycosylation), and clonal variation following genomic integration. Modified and degraded polypeptide products can be incorporated into fibers without adversely affecting the utility of the fibers depending on their intended use.

TABLE 6 Full length block copolymer silk constructs with alpha mating factor, 4X repeat domains, and 3X FLAG domains. Western Results (1 = no band Amino acid Nucleotide 2 = weak band Construct ID R/N/C SEQ ID NO SEQ ID NO: 3 = strong band) 4x 269 R 1411 1440 2 4x 340 R 1412 1441 3 4x 153 R 1413 1442 3 4x 291 R 1414 1443 3 4x 350 R 1415 1444 3 4x 228 R 1416 1445 2 4x 159 R 1417 1446 3 4x 295 R 1418 1447 3 4x 355 R 1419 1448 3 4x 241 R 1420 1449 3 4x 178 R 1421 1450 3 4x 305 R 1422 1451 3 4x 362 R 1423 1452 2 4x 283 R 1424 1453 3 4x 183 R 1425 1454 3 4x 316 R 1426 1455 3 2x 362 + 2x 370 R 1509 2802 3 4x 302 R 1427 1456 3 4x 209 R 1428 1457 3 2x 183 + 2x 320 R 1511 1510 2 4x 403 R 1430 1459 3 4x 330 R 1431 1460 2 4x 222 R 1432 1461 3 4x 326 R 1433 1462 2 4x 429 R 1434 1463 3 4x 384 R 1435 1464 1 4x 239 R 1436 1465 2 4x 333 R 1437 1466 3 4x 457 R 1438 1467 2 4x 406 R 1439 1468 2

Example 7 Expression of 18B from Bacillus subtilis

An E. coli/B. subtilis shuttle and expression plasmid is first constructed. The polynucleotide encoding 18B is transferred, using a Gibson reaction, to plasmid pBE-S (Takara Bio Inc.). Plasmid pBE-S(SEQ ID NO: 1512) is amplified using primers BES-F (5′-AAGACGATGACGATAAGGACTATAAAGATGATGACGACAAATAATGCGGTAGTT TATCAC-3′) (SEQ ID NO: 2831) and BES-R (5′-CCAGCGCCTGGACCGTAACCCGGCCGCAGCCTGCGCAGACATGTTGCTGAACGC CATCGT-3′) (SEQ ID NO: 2832) in a PCR reaction. The reaction mixture consists of 1 μl of 10 μM BES-F, 1 μl of 10 μM BES-R, 0.5 μg of pBE-S DNA (in 1 μl volume), 22 μl of deionized H2O, and 25 μl of Phusion High-Fidelity PCR Master Mix (NEB catalog M0531S). The mixture is thermocycled according to the following program:

-   -   1) Denature for 5 minutes at 95° C.     -   2) Denature for 30 seconds at 95° C.     -   3) Anneal for 30 seconds at 55° C.     -   4) Extend for 6 minutes at 72° C.     -   5) Repeat steps 2-4 for 29 additional cycles     -   6) Perform a final extension for 5 minutes at 72° C.

The product is subjected to gel electrophoresis, and the product of approximately 6000 bp is isolated, then extracted using a Zymoclean Gel DNA Recovery Kit (Zymo Research) according to the manufacturer's instructions. The polynucleotide encoding 18B is isolated by digestion of 18B in the KA assembly vector using restriction enzyme BtgZI, followed by gel electrophoresis, fragment isolation, and gel extraction. The pBE-S and 18B fragments are joined together using Gibson Assembly Master Mix (New England Biolabs) according to the manufacturer's instructions, and the resulting plasmid transformed into E. coli using standard techniques for subsequent clonal isolation, DNA amplification, and DNA purification. The resulting plasmid, pBE-S-18B (SEQ ID NO: 1513), is then diversified by insertion of various signal peptides (the “SP DNA mixture”) according to the manufacturer's instructions. A mixture of pBE-S-18B plasmids containing different secretion signal peptides is then transformed into B. subtilis strain RIK1285 according to the manufacturer's instructions. 96 of the resulting colonies are incubated in TY medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) for 48 hours, at which point the cells are pelleted and the supernatant is analyzed by western blot for expression of the 18B polypeptide.

Example 8 Expression of 18B from Chlamydomonas reinhardtii

An E. coli vector bearing an excisable C. reinhardtii expression cassette, pChlamy (SEQ ID NO: 1514), is first constructed using commercial DNA synthesis and standard techniques. The cassette is described in detail in Rasala, B. A., Robust expression and secretion of Xylanasel in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide, PLoS One, 7:8 (2012). The polypeptide encoding 18B, a 3×FLAG tag, and a stop codon is reverse translated using the codon preference of C. reinhardtii (available, for example, at http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=3055) and synthesized using commercial synthesis. During synthesis, flanking BbsI sites are included to allow release of the 18B-3×FLAG polynucleotide. The polynucleotide resulting from PCR amplification of the pChlamy plasmid using primers designed to generate a linear fragment including the entire plasmid sequence except 5′-ATGTTTTAA-3′ and also including 40 bp of homology to the 18B-3×FLAG coding sequence on each end is joined with the 18B-3×FLAG polynucleotide liberated by digestion with BbsI using a Gibson reaction, and transformed into E. coli for clonal selection, DNA amplification, and plasmid isolation. The resulting plasmid is digested with BsaI to release the 18B expression cassette, which is isolated by gel purification. The digested fragment is electroporated into strain cc3395, which is then selected on 15 μg/ml zeocin. Several clones are grown up in liquid culture, the cells pelleted by centrifugation, and the supernatant analyzed by western blot for protein expression.

Example 9 Additional Silk and Silk-Like Sequences

Additional silk and silk-like sequences and partial sequences were obtained from NCBI's sequence database by search for the term “silk” while excluding “spidroin” “bombyx” and “latrodectus”. A subset of the resulting nucleotide sequences were translated into amino acid sequences, then curated to remove repeated sequences. Short sequences, generally less than 200-500 amino acids long, were removed. Further, primary sequences for select polypeptides known to form structural elements were obtained from public databases. Amino acid sequences so obtained, in addition to the sequences described in Example 1, were used to search for additional silk and silk-like sequences by homology. Resulting silk and silk-like sequences were curated, then partitioned into repetitive and non-repetitive regions.

Repetitive polypeptide sequences (repeat (R) sequences) were selected from each silk sequence and include SEQ ID NOs: 2157-2690 (SEQ ID NOs: 2157-2334 are nucleotide sequences, SEQ ID NOs: 2335-2512 are nucleotide sequences with flanking sequences for cloning, and SEQ ID NOs: 2513-2690 are amino acid sequences). Some of the R sequences have been altered, e.g., by addition of a serine to the C terminus to avoid terminating the sequence with an F, Y, W, C, H, N, M, or D amino acid. This allows for incorporation into the vector system described above. Incomplete blocks may also have been altered by incorporation of segments from a homologous sequence from another block.

Non-repetitive N terminal domain sequences (N sequences) and C terminal domain sequences (C sequences) were also selected from some silk and silk-like sequences (SEQ ID NOs: 2157-2690). The N terminal domain sequences were altered by removal of the leading signal sequence and, if not already present, addition of an N-terminal glycine residue. In some cases, the N and/or C domains were not separated from the R sequence(s) before further processing. R, N, and C amino acid sequences were reverse translated to nucleotide sequences as described in Example 2. The resulting nucleotide sequences were flanked with the following sequences during synthesis to enable cloning:

5′-GAAGACTTAA-SILK-GGTACGTCTTC-3′ (SEQ ID NOS 2833 and 2826) where “SILK” is a polynucleotide sequence selected according to the teachings above.

Resulting linear DNA was digested with BbsI and ligated into vector RM747 (SEQ ID NO: 2696) which had been digested with BsmBI to release a dummy insert. Ligated material was transformed into E. coli for clonal isolation, DNA amplification, and sequence verification using standard methods. Resulting plasmids were digested with BsaI and BbsI, and the fragment encoding a silk or silk-like polypeptide isolated by gel electrophoresis, fragment excision, and gel extraction. The fragment was subsequently ligated into Expression Vector RM1007 (SEQ ID NO: 2707) which had been digested with BsmBI and treated with Calf Intestinal Alkaline Phosphatase. Ligated material was transformed into E. coli for clonal isolation, DNA amplification, and sequence verification using standard methods.

Expression vectors containing R, N, and/or C sequences were transformed into Pichia (Komagataella) pastoris (strain RMs71, described in Example 3) using the PEG method (Cregg, J. M. et al., DNA-mediated transformation, Methods Mol. Biol., 389, pg. 27-42 (2007)). The expression vector consisted of a targeting region and promoter (pGAP), a dominant resistance marker (nat-conferring resistance to nourseothricin), a secretion signal (alpha mating factor leader and pro sequence), a C-terminal 3×FLAG epitope, and a terminator (pAOX1 pA signal).

Transformants were plated on Yeast Extract Peptone Dextrose Medium (YPD) agar plates containing 25 μg/ml nourseothricin and incubated for 48 hours at 30° C. Two clones from each transformation were inoculated into 400 μl of Buffered Glycerol-complex Medium (BMGY) in a 96-well square-well block, and incubated for 48 hours at 30° C. with agitation at 1000 rpm. Cells were pelleted via centrifugation, and the supernatant was recovered for analysis of block copolymer polypeptide content via western blot analysis of the 3×FLAG epitope.

Successful polypeptide expression and secretion was judged by western blot. Each western lane was scored as 1: No band 2: Moderate band or 3: Intense band. The higher of the two scores for each clone was recorded. Representative western blot data are shown in FIG. 14. A complete listing of all R, N, and C sequences tested along with western blot results is shown in Table 7. Silk and silk-like block copolymer polypeptides from numerous species expressed successfully, encompassing diverse species and diverse polypeptide structures.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention.

TABLE 7 Additional silk polypeptide sequences Nucleotide Western Results with flanking (1 = no band Construct N/C/R Nucleotide sequences Amino Acid 2 = weak band # Species sequence SEQ ID NO SEQ ID NO: SEQ ID NO: 3 = strong band) 463 Ceratitis capitata R 2157 2335 2513 no data 464 Archimantis monstrosa NRC 2158 2336 2514 no data 465 Archimantis monstrosa NRC 2159 2337 2515 no data 466 Pseudomantis albofimbriata NRC 2160 2338 2516 1 467 Pseudomantis albofimbriata NRC 2161 2339 2517 no data 468 Tenodera australasiae NRC 2162 2340 2518 2 469 Tenodera australasiae NRC 2163 2341 2519 no data 470 Hydropsyche angustipennis R 2164 2342 2520 1 471 Hydropsyche angustipennis R 2165 2343 2521 no data 472 Hydropsyche angustipennis N 2166 2344 2522 no data 473 Hydropsyche angustipennis C 2167 2345 2523 no data 474 Hydropsyche sp. T20 R 2168 2346 2524 no data 475 Rhyacophila obliterata R 2169 2347 2525 no data 476 Rhyacophila obliterata R 2170 2348 2526 no data 477 Rhyacophila obliterata C 2171 2349 2527 no data 478 Rhyacophila obliterata N 2172 2350 2528 no data 479 Limnephilus decipiens R 2173 2351 2529 no data 480 Chironomus pallidivittatus NRC 2174 2352 2530 no data 481 Chironomus pallidivittatus R 2175 2353 2531 3 482 Chironomus pallidivittatus R 2176 2354 2532 no data 483 Chironomus thummi R 2177 2355 2533 3 484 Stenopsyche marmorata R 2178 2356 2534 1 485 Mallada signata R 2179 2357 2535 3 486 Mallada signata N 2180 2358 2536 3 487 Mallada signata C 2181 2359 2537 3 488 Mallada signata R 2182 2360 2538 3 489 Mallada signata R 2183 2361 2539 3 490 Mallada signata N 2184 2362 2540 no data 491 Mallada signata C 2185 2363 2541 3 492 Mallada signata R 2186 2364 2542 no data 493 Haploembia solieri R 2187 2365 2543 no data 494 Culex quinquefasciatus R 2188 2366 2544 no data 495 Culex quinquefasciatus R 2189 2367 2545 1 496 Oecophylla smaragdina NRC 2190 2368 2546 no data 497 Oecophylla smaragdina NRC 2191 2369 2547 no data 498 Oecophylla smaragdina NRC 2192 2370 2548 no data 499 Oecophylla smaragdina NRC 2193 2371 2549 2 500 Myrmecia forficata NRC 2194 2372 2550 no data 501 Myrmecia forficata NRC 2195 2373 2551 2 502 Myrmecia forficata NRC 2196 2374 2552 no data 503 Myrmecia forficata NRC 2197 2375 2553 no data 504 Bombus terrestris NRC 2198 2376 2554 no data 505 Bombus terrestris NRC 2199 2377 2555 no data 506 Bombus terrestris NRC 2200 2378 2556 no data 507 Bombus terrestris NRC 2201 2379 2557 3 508 Bombus terrestris NRC 2202 2380 2558 no data 509 Vespa simillima xanthoptera R 2203 2381 2559 3 510 Vespa simillima xanthoptera R 2204 2382 2560 2 511 Vespa simillima xanthoptera R 2205 2383 2561 no data 512 Vespa simillima xanthoptera NRC 2206 2384 2562 3 513 Vespa simillima xanthoptera NRC 2207 2385 2563 no data 514 Vespa simillima xanthoptera NRC 2208 2386 2564 no data 515 Apis mellifera NRC 2209 2387 2565 no data 516 Apis mellifera NRC 2210 2388 2566 no data 517 Apis mellifera NRC 2211 2389 2567 no data 518 Apis mellifera NRC 2212 2390 2568 no data 519 Cotesia glomerata R 2213 2391 2569 no data 520 Aposthonia gurneyi R 2214 2392 2570 no data 521 Hilara sp. TDS-2007 R 2215 2393 2571 no data 522 Hilara sp. TDS-2007 R 2216 2394 2572 1 523 Hilara sp. TDS-2007 R 2217 2395 2573 no data 524 Apotrechus illawarra NRC 2218 2396 2574 no data 525 Apotrechus illawarra R 2219 2397 2575 3 526 Cricula trifenestrata R 2220 2398 2576 2 527 Antheraea yamamai N 2221 2399 2577 no data 528 Antheraea yamamai C 2222 2400 2578 no data 529 Antheraea yamamai R 2223 2401 2579 no data 530 Antheraea yamamai R 2224 2402 2580 no data 531 Antheraea yamamai R 2225 2403 2581 no data 532 Antheraea yamamai R 2226 2404 2582 no data 533 Antheraea pernyi N 2227 2405 2583 no data 534 Antheraea pernyi C 2228 2406 2584 no data 535 Antheraea pernyi R 2229 2407 2585 no data 536 Antheraea pernyi R 2230 2408 2586 2 537 Antheraea mylitta R 2231 2409 2587 2 538 Saturnia japonica N 2232 2410 2588 2 539 Saturnia japonica R 2233 2411 2589 no data 540 Saturnia japonica R 2234 2412 2590 2 541 Saturnia japonica R 2235 2413 2591 no data 542 Rhodinia fugax N 2236 2414 2592 no data 543 Rhodinia fugax R 2237 2415 2593 no data 544 Rhodinia fugax R 2238 2416 2594 no data 545 Rhodinia fugax R 2239 2417 2595 no data 546 Rhodinia fugax R 2240 2418 2596 no data 547 Galleria mellonella N 2241 2419 2597 3 548 Galleria mellonella C 2242 2420 2598 2 549 Galleria mellonella R 2243 2421 2599 no data 550 Galleria mellonella R 2244 2422 2600 no data 551 Bombyx mori N 2245 2423 2601 3 552 Bombyx mori C 2246 2424 2602 2 553 Bombyx mori R 2247 2425 2603 no data 554 Bombyx mori R 2248 2426 2604 2 555 Bombyx mori R 2249 2427 2605 no data 556 Anagasta kuehniella N 2250 2428 2606 no data 557 Anagasta kuehniella C 2251 2429 2607 no data 558 Anagasta kuehniella R 2252 2430 2608 no data 559 Anagasta kuehniella R 2253 2431 2609 no data 560 Antheraea pernyi R 2254 2432 2610 2 561 Antheraea pernyi C 2255 2433 2611 no data 562 Bacillus cereus R 2256 2434 2612 2 563 Bacillus cereus R 2257 2435 2613 3 564 Bacillus cereus R 2258 2436 2614 2 565 Bacillus thuringiensis R 2259 2437 2615 2 566 Bacillus licheniformis R 2260 2438 2616 2 567 Bacillus licheniformis R 2261 2439 2617 1 568 Neospora caninum R 2262 2440 2618 no data 569 Danio rerio R 2263 2441 2619 no data 570 Danio rerio R 2264 2442 2620 no data 571 Danio rerio R 2265 2443 2621 no data 572 Atta cephalotes R 2266 2444 2622 2 573 Ureaplasma urealyticum R 2267 2445 2623 1 574 Bombus terrestris R 2268 2446 2624 no data 575 Bombus terrestris R 2269 2447 2625 no data 576 Bombus impatiens R 2270 2448 2626 no data 577 Bombus impatiens R 2271 2449 2627 no data 578 Bombus impatiens R 2272 2450 2628 no data 579 Bombus impatiens R 2273 2451 2629 no data 580 Bombus impatiens R 2274 2452 2630 1 581 Drosophila yakuba R 2275 2453 2631 no data 582 Drosophila yakuba R 2276 2454 2632 2 583 Pseudomonas syringae R 2277 2455 2633 no data 584 Phytophthora infestans R 2278 2456 2634 no data 585 Phytophthora sojae R 2279 2457 2635 no data 586 Polysphondylium pallidum R 2280 2458 2636 no data 587 Rhipicephalus pulchellus R 2281 2459 2637 no data 588 Culex quinquefasciatus R 2282 2460 2638 no data 589 Tribolium castaneum R 2283 2461 2639 no data 590 Tribolium castaneum R 2284 2462 2640 no data 591 Streptococcus pyogenes R 2285 2463 2641 2 592 Candidatus Microthrix parvicella R 2286 2464 2642 no data 593 Amphimedon queenslandica R 2287 2465 2643 no data 594 Acyrthosiphon pisum R 2288 2466 2644 no data 595 Acyrthosiphon pisum R 2289 2467 2645 no data 596 Caenorhabditis brenneri R 2290 2468 2646 no data 597 Caenorhabditis brenneri R 2291 2469 2647 2 598 Burkholderia pseudomallei R 2292 2470 2648 no data 599 Mustela putorius furo R 2293 2471 2649 3 600 Candida parapsilosis R 2294 2472 2650 no data 601 Candida parapsilosis R 2295 2473 2651 no data 602 Candida parapsilosis R 2296 2474 2652 no data 603 Paenibacillus sp R 2297 2475 2653 no data 604 Xenopus (Silurana) tropicalis R 2298 2476 2654 no data 605 Xenopus (Silurana) tropicalis R 2299 2477 2655 2 606 Anopheles darlingi R 2300 2478 2656 no data 607 Anopheles darlingi R 2301 2479 2657 no data 608 Drosophila melanogaster R 2302 2480 2658 2 609 Drosophila melanogaster R 2303 2481 2659 no data 610 Synechococcus phage P60 R 2304 2482 2660 no data 611 Amblyomma variegatum R 2305 2483 2661 no data 612 Kazachstania naganishii R 2306 2484 2662 no data 613 Drosophila ananassae R 2307 2485 2663 no data 614 Tetrapisispora blattae R 2308 2486 2664 2 615 Tetrapisispora blattae R 2309 2487 2665 no data 616 Monodelphis domestica R 2310 2488 2666 no data 617 Amblyomma variegatum R 2311 2489 2667 no data 618 Amblyomma variegatum R 2312 2490 2668 no data 619 Latrodectus hesperus R 2313 2491 2669 no data 620 Danaus plexippus R 2314 2492 2670 no data 621 Encephalitozoon intestinalis R 2315 2493 2671 no data 622 Encephalitozoon intestinalis R 2316 2494 2672 no data 623 Psychromonas ingrahamii R 2317 2495 2673 no data 624 Drosophila melanogaster R 2318 2496 2674 no data 625 Chironomus tentans R 2319 2497 2675 no data 626 Acyrthosiphon pisum R 2320 2498 2676 1 627 Megachile rotundata R 2321 2499 2677 no data 628 Megachile rotundata R 2322 2500 2678 no data 629 Acyrthosiphon pisum R 2323 2501 2679 no data 630 Pseudomonas syringae R 2324 2502 2680 no data 631 Nematostella vectensis R 2325 2503 2681 no data 632 Dasypus novemcinctus R 2326 2504 2682 3 633 Trichoderma harzianum R 2327 2505 2683 3 634 Nematostella vectensis R 2328 2506 2684 no data 635 Nematostella vectensis R 2329 2507 2685 no data 636 Caenorhabditis elegans R 2330 2508 2686 no data 637 Leishmania mexicana R 2331 2509 2687 no data 638 Chelonia mydas R 2332 2510 2688 2 639 Nasonia vitripennis R 2333 2511 2689 no data 640 Euprymna scolopes NRC 2334 2512 2690 no data

Example 10 Circularly Permuted Variants of Argiope bruennichi MaSp2 Polypeptides

The 6 repeat blocks (block co-polymer) from Argiope bruennichi MaSp2 identified in Example 5 were circularly permuted by approximately 90 degrees (by moving ˜1.5 blocks from the end of the six blocks to the beginning), then divided into 2 R sequences consisting of ˜3 blocks each, RM2398 (SEQ ID NO: 2708) and RM2399 (SEQ ID NO: 2709). These 3-block sequences were subsequently used to generate 6-block sequences rotated by ˜90 and ˜270 degrees from the original 6-block sequence, and existing 3-block sequences (RM409 and RM410) were used to generate a 6-block sequence rotated by ˜180 degrees. Each 6-block sequence was then assembled into 18-block sequences. The assembly process and rotated sequences are depicted in FIG. 15.

To generate RM2398 and RM2399, plasmid RM439 (SEQ ID NO: 467) was amplified by PCR using either primers RM2398F (5′-CTAAGAGGTCTCACAGGTAGTCAAGGACCTGGTTCAGG-3′) (SEQ ID NO: 2834) and RM2398R (5′-TTCAGTGGTCTCTACCTTGTTGTCCTCCAGATCCAG-3′) (SEQ ID NO: 2835) or RM2399F (5′-CTAAGAGGTCTCACAGGTCCTGGAGGTCAGGGTCCAT-3′) (SEQ ID NO: 2836) and RM2399R (5′-TTCAGTGGTCTCTACCTGGTCCCTGTTGACCAGCACCAGGA-3′) (SEQ ID NO: 2837). Each reaction consisted of 12.5 μL 2×KOD Extreme Buffer, 0.25 μl KOD Extreme Hot Start Polymerase, 0.5 μl 10 μM Fwd oligo, 0.5 μl 10 μM Rev oligo, 5 ng template DNA (RM439), 0.5 μl of 10 mM dNTPs, and ddH2O added to final volume of 25 Each reaction was then thermocycled according to the program:

-   -   1. Denature at 94° C. for 5 minutes     -   2. Denature at 94° C. for 30 seconds     -   3. Anneal at 55° C. for 30 seconds     -   4. Extend at 72° C. for 60 seconds     -   5. Repeat steps 2-4 for 29 additional cycles     -   6. Final extension at 72° C. for 5 minutes         Resulting linear DNA was digested with BsaI and ligated into         assembly vectors RM2086 (SEQ ID NO: 2693) and RM2089 (SEQ ID         NO: 2695) that had been digested with BsmBI. Ligated material         was transformed into E. coli for clonal isolation, DNA         amplification, and sequence verification using standard methods.         Using the 2ab assembly process described in Example 5 (with         minor modifications to the assembly vectors to shift the BtgZI         cut sites further away from the silk sequences), the 3-block         fragments were assembled into two different 6-block fragments,         one with RM2398 proceeding RM2399 (producing RM2452-SEQ ID NO:         2710), and one with RM2399 proceeding RM2398 (producing         RM2454-SEQ ID NO: 2712). Additionally, RM409 (SEQ ID NO 463) and         RM410 (SEQ ID NO 464) were digested out of the assembly vector         RM396 with BbsI and BsaI, and ligated into vector RM2105 (SEQ ID         NO: 2691) that had been digested with BbsI and BsaI and treated         with Calf Intestinal Alkaline Phosphatase. Ligated material was         transformed into E. coli for clonal isolation, DNA         amplification, and sequence verification using standard methods.         The resulting plasmids were subsequently digested with AscI and         SbfI and the fragments encoding a silk isolated by gel         electrophoresis, fragment excision, and gel extraction. The         fragments were subsequently ligated into assembly vectors RM2086         and RM2089 that had been digested with AscI and SbfI. Ligated         material was transformed into E. coli for clonal isolation, DNA         amplification, and sequence verification using standard methods.         Using 2ab assembly, a 6-block fragment consisting of RM410         proceeding RM409 was generated (producing RM2456-SEQ ID NO:         2711). RM2452, RM2454, and RM2456 were digested from assembly         vector RM2081 (SEQ ID NO: 2692) with AscI and SbfI, and ligated         into assembly vectors RM2088 and RM2089 that had been digested         with AscI and SbfI. Ligated material was transformed into E.         coli for clonal isolation, DNA amplification, and sequence         verification using standard methods. Using 2ab assembly,         18-block sequences were generated from each of the three 6-block         fragments, resulting in sequences RM2462 (SEQ ID NO: 2713),         RM2464 (SEQ ID NO: 2715), and RM2466 (SEQ ID NO: 2714). Each of         the 6-block and 18-block sequences was then digested from the         assembly vector using BsaI and BbsI, and the fragments encoding         a silk isolated by gel electrophoresis, fragment excision, and         gel extraction. The fragments were subsequently ligated         expression vector RM1007 (SEQ ID NO: 2707) that had been         digested with BsmBI and treated with Calf Intestinal Alkaline         Phosphatase. Ligated material was transformed into E. coli for         clonal isolation, DNA amplification, and sequence verification         using standard methods. Resulting plasmids were linearized with         BsaI and used to transform Pichia (Komagataella) pastoris         (strain RMs71, described in Example 3) using the PEG method         (Cregg, J. M. et al., DNA-mediated transformation, Methods Mol.         Biol., 389, pg. 27-42 (2007)). Transformants were plated on         Yeast Extract Peptone Dextrose Medium (YPD) agar plates         containing 25 μg/ml nourseothricin and incubated for 48 hours at         30° C. Two clones from each transformation were inoculated into         400 μl of Buffered Glycerol-complex Medium (BMGY) in a 96-well         square-well block, and incubated for 48 hours at 30° C. with         agitation at 1000 rpm. Cells were pelleted via centrifugation,         and the supernatant was recovered for analysis of silk         polypeptide content via western blot analysis of the 3×FLAG         epitope. Western blot data for a representative clone of each         polypeptide is shown in FIG. 16. Expression and secretion of         each of the circularly permuted polypeptides appears comparable         to its un-rotated counterpart. This suggests that any number of         starting positions can be selected for identifying blocks in         repeated silk or silk-like polypeptides without consequence on         the expression or secretion of polypeptides composed of those         blocks.

Example 11 Changing Expression of an Argiope bruennichi MaSp2 Polynucleotide Through Control of Copy Number and Promoter Strength

The degree of transcription of an exogenously introduced polynucleotide is known to affect the amount of polypeptide produced (see e.g. Liu, H., et al., Direct evaluation of the effect of gene dosage on secretion of protein from yeast Pichia pastoris by expressing EGFP, J. Microbiol. Biotechnol., 24:2, pg. 144-151 (2014); and Hohenblum, H., et al., Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris, Biotechnol. Bioeng., 85:4, pg. 367-375 (2004)). In Pichia (Komagataella) pastoris, the degree of transcription is commonly controlled either by increasing the number of copies of a polynucleotide that are integrated into the host genome or by selecting an appropriate promoter to drive transcription (see e.g. Hartner, F. S., et al., Promoter library designed for fine-tuned gene expression in Pichia pastoris, Nucleic Acids Res., 36:12 (2008); Zhang, A. L., et al., Recent advances on the GAP promoter derived expression system of Pichia pastoris, Mol. Biol. Rep., 36:6, pg. 1611-1619 (2009); Ruth, C., et al., Variable production windows for porcine trypsinogen employing synthetic inducible promoter variants in Pichia pastoris, Syst. Synth. Biol., 4:3, pg. 181-191 (2010); Stadlmayr, G., et al., Identification and characterisation of novel Pichia pastoris promoters for heterologous protein production, J. Biotechnol., 150:4, pg. 519-529 (2010)). A relatively recent addition to the set of promoters used for heterologous protein expression is pGCW14 (Liang, S., Identification and characterization of P GCW14: a novel, strong constitutive promoter of Pichia pastoris, Biotechnol. Lett. 35:11, pg. 1865-1871 (2013)), which is reported to be 5-10 times stronger than pGAP. To validate that the expression and secretion of silk and silk-like polypeptides can also be influenced by copy number, strains containing 1, 3, or 4 copies of pGAP driving expression of 18B (described in Example 5) and strains containing 1, 2, 3, or 4 copies of pGCW14 driving expression of 18B were generated and tested. The strains are described in Table 8.

TABLE 8 Strains with multiple polynucleotide sequences or different promoters Newly Strain Derived incorporated ID Description From sequence(s) Selection RMs126 1x pGAP 18B GS115 RM439 in RM630 Minimal (NRRL Dextrose Y15851) RMs127 3x pGAP 18B RMs126 RM439 in RM632 nourseothricin, and RM633 hygromycin B RMs134 4x pGAP 18B RMs127 RM439 in RM631 G418 RMs133 1x pGCW14 18B GS115 RM439 in RM812 Minimal (NRRL Dextrose Y15851) RMs138 2x pGCW14 18B RMs133 RM439 in RM814 nourseothricin RMs143 3x pGCW14 18B RMs138 RM439 in RM815 hygromycin B RMs152 4x pGCW14 18B RMs143 RM439 in RM837 G418

The polynucleotide sequence encoding alpha mating factor+18B+3×FLAG tag was digested from the plasmid described in Example 5 (RM468, SEQ ID NO: 1401, with RM439, SEQ ID NO: 467 cloned in) using restriction enzyme AscI and SbfI. The fragment encoding alpha mating factor+18B+3×FLAG tag was isolated by gel electrophoresis, fragment excision, and gel extraction. The resulting linear DNA was ligated into expression vectors RM630 (SEQ ID NO: 2697), RM631 (SEQ ID NO: 2698), RM632 (SEQ ID NO: 2699), RM633 (SEQ ID NO: 2700), RM812 (SEQ ID N: 2701), RM837 (SEQ ID NO: 2702), RM814 (SEQ ID N: 2703), and RM815 (SEQ ID NO: 2704) that had been digested with AscI and SbfI. Key attributes of the expression vectors are summarized in Table 9, and sequences include SEQ ID NOs: 2691-2707. Ligated material was transformed into E. coli for clonal isolation, DNA amplification, and sequence verification using standard methods.

TABLE 9 Additional vectors SEQ Vector ID ID NO: Description RM2105 2691 Vector for receiving silks before transfer to some assembly vectors. p15a origin, gentamycin resistance RM2081 2692 CK assembly vector with revised BtgZI targeting, p15a origin RM2086 2693 CA assembly vector with revised BtgZI targeting, p15a origin RM2088 2694 KA assembly vector with revised BtgZI targeting, p15a origin RM2089 2695 AK assembly vector with revised BtgZI targeting, p15a origin RM747 2696 Vector for receiving silks before transfer to some assembly vectors. p15a origin, gentamycin resistance RM630 2697 Expression vector. Integrates into HIS4 locus. pGAP promoter. RM631 2698 Expression vector. Integrates into AOX2 locus. pGAP promoter. Confers G418 resistance RM632 2699 Expression vector. Integrates into HSP82 locus. pGAP promoter. Confers nourseothricin resistance RM633 2700 Expression vector. Integrates into TEF1 locus. pGAP promoter. Confers hygromycin B resistance RM812 2701 Expression vector. Integrates into HIS4 locus. pGCW14 promoter. RM837 2702 Expression vector. Integrates into AOX2 locus. pGCW14 promoter. Confers G418 resistance RM814 2703 Expression vector. Integrates into HSP82 locus. pGCW14 promoter. Confers nourseothricin resistance RM815 2704 Expression vector. Integrates into TEF1 locus. pGCW14 promoter. Confers hygromycin B resistance RM785 2705 Expression vector. Integrates into pGAP locus. pGAP promoter. Confers nourseothricin resistance RM793 2706 Expression vector. Integrates into HSP82 locus. pGAP promoter. Confers nourseothricin resistance RM1007 2707 Expression vector. Integrates into pGAP locus. pGAP promoter. Confers nourseothricin resistance

The polynucleotide encoding 18B in expression vector RM630 was linearized with BsaI and transformed into Pichia (Komagataella) pastoris (strain GS115-NRRL Y15851) using the PEG method (Cregg, J. M. et al., DNA-mediated transformation, Methods Mol. Biol., 389, pg. 27-42 (2007)). Transformants were plated on Minimal Dextrose (MD) agar plates (no added amino acids) and incubated for 48 hours at 30° C. This resulted in creation of strain RMs126, 1×pGAP 18B.

RMs126 was subsequently co-transformed with the polynucleotide encoding 18B in expression vectors RM632 and RM633 (linearized with BsaI) using the electroporation method (Wu., S., and Letchworth, G. J., High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol, Biotechniques, 36:1, pg. 152-154 (2004)). Transformants were plated on Yeast Extract Peptone Dextrose Medium (YPD) agar plates containing 25 μg/ml nourseothricin and 100 ug/ml hygromycin B and incubated for 48 hours at 30° C. This resulted in creation of strain RMs127, 3×pGAP 18B.

RMs127 was subsequently transformed with the polynucleotide encoding 18B in expression vector RM631 (linearized with BsaI) using the PEG method. Transformants were plated on Yeast Extract Peptone Dextrose Medium (YPD) agar plates containing 300 μg/ml G418 and incubated for 48 hours at 30° C. This resulted in creation of strain RMs134, 4×pGAP 18B.

To generate strains RMs133, RMs138, RMs143, and RMs152 (1×, 2×, 3×, and 4× p754 18B, respectively), strain GS115 (NRRL Y15851) was serially transformed with the polynucleotide encoding 18B in expression vectors RM812, RM814, RM815, and RM837 (after linearizing with BsaI) using the PEG method.

A clone of each strain was inoculated into 400 μl of Buffered Glycerol-complex Medium (BMGY) in a 96-well square-well block, and incubated for 48 hours at 30° C. with agitation at 1000 rpm. Cells were pelleted via centrifugation, and the supernatant was recovered for analysis of block copolymer polypeptide content via western blot analysis of the 3×FLAG epitope. Western blot data for a representative clone of each polypeptide is shown in FIG. 16. Increasing band intensities suggest that higher transcription resulted in the expression and secretion of additional block copolymer polypeptide, confirming that the strategy of increasing transcription functions on block copolymer based on silk and silk-like polypeptide repeat units.

Example 12 Comparing Expression and Secretion of Single R Domains to Homopolymers of R Domains

Additional selected R domains from SEQ ID NOs: 1-1398 that expressed and secreted well were concatenated into 4 to 6× repeat domains using the 2ab assembly (described in Example 5). Additionally, 2ab assembly was used to concatenate a 12B sequence with an 18B sequence (from Example 5), resulting in a 30B sequence. The resulting products were transferred into an expression vector, such that each silk sequence is flanked by alpha mating domain on the 5′ end and a 3×FLAG domain on the 3′ end and driven by a pGAP promoter. The sequences generated are described in Table 10, and the sequences include SEQ ID NOs: 2734-2748.

TABLE 10 Additional full-length block copolymer constructs with alpha mating factor, multiple repeat domains, and 3X FLAG domains Predicted DNA Amino acid Molecular (with alpha (with alpha Weight of mating factor mating factor Secreted and 3x FLAG) and 3x FLAG) Product Expression Construct ID SEQ ID NO: SEQ ID NO: (kDa) Vector 4x 438 2724 2734 63.4 RM652  4x 412 2725 2735 77.1 RM1007 6x 415 2726 2736 75.9 RM1007 5x 317 2727 2737 70.1 RM1007 5x 303 2728 2738 62.0 RM1007 5x 310 2729 2739 62.7 RM1007 4x 301 2730 2740 47.3 RM793  4x 410 2731 2741 52.3 RM793  4x 451 2732 2742 57.7 RM793  4x 161 2733 2743 44.9 RM785  RM2361 (30B) 2744 2745 135.1 RM1007 RM411 (6B) 2746 2749 29.5 RM1007 RM434 (12B) 2747 2750 55.9 RM1007 RM439 (18B) 2748 2751 82.31 RM1007

The block copolymer expression vectors were then transformed into Pichia (Komagataella) pastoris (strain RMs71, described in Example 3) using the PEG method (Cregg, J. M. et al., DNA-mediated transformation, Methods Mol. Biol., 389, pg. 27-42 (2007)). Transformants were plated on YPD agar plates containing 25 μg/ml nourseothricin and incubated for 48 hours at 30° C. Three clones from each transformation were picked into 400 μl of BMGY in a 96-well square-well block, and incubated for 48 hours at 30° C. with agitation at 1000 rpm. Cells were pelleted via centrifugation, and the supernatant was recovered for analysis of silk polypeptide content via western blot. A representative clone for each block copolymer construct, as well as the 1×R domain counterpart and 4×R domain constructs from Example 6, are show in FIG. 16. As observed in Example 6, streakiness and multiple bands are evident on the western blot. While the specific source of these variations has not been identified, they are generally consistent with typically observed phenomena, including polypeptide degradation and post-translational modification (e.g. glycosylation). Further, the band intensity of 4-6×R domain polypeptides appears to be weaker than the corresponding 1×R domain constructs. This is also evident in the 6B, 12B, 18B, and 30B series of Argiope bruennichi MaSp2 polypeptides. This suggests that longer block copolymers comprising silk repeat sequences are generally less well expressed and secreted than shorter block copolymer sequences comprising the same or different repeat sequences.

Example 13 Measuring Productivity of Strains Expressing and Secreting Silks

Table 11 lists the volumetric and specific productivities of strains expressing the polypeptides described in Example 10, Example 11, and Example 12.

TABLE 11 Productivity of strains producing silk polypeptides Volumetric Volumetric Specific Specific productivity productivity productivity productivity (mg silk/liter/ error (SD, (mg silk/g error (SD, Construct ID hour) n = 3) DCW/hour) n = 3) 1x 159 5.82 0.29 1.70 0.18 1x 295 5.47 0.27 1.64 0.17 1x 179 3.90 0.92 1.16 0.33 1x 340 4.94 0.05 1.45 0.10 1x 283 7.57 0.48 2.28 0.26 1x 301 3.75 0.27 1.11 0.14 1x 410 4.31 0.28 1.34 0.03 1x 451 6.69 0.36 2.16 0.11 1x 161 4.55 0.09 1.45 0.22 4x 478 1.08 0.17 0.34 0.09 4x 340 4.91 0.59 1.58 0.41 RM2464 (18B, 19.13 0.14 5.25 0.64 270 degree rotation) RM2466 (18B, 15.70 0.60 4.48 0.61 180 degree rotation) RM439 (18B, 19.22 0.84 5.53 0.68 unrotated) RM2452 (6B, 9.28 0.07 2.63 0.15 90 degree rotation) RM2454 (6B, 10.76 0.40 3.18 0.22 180 degree rotation) RM2456 (6B, 10.21 0.23 2.99 0.22 180 degree rotation) RM2462 (18B, 15.25 0.56 4.69 0.33 90 degree rotation) 1x 412 2.95 0.53 0.96 0.22 1x 415 7.67 0.69 2.18 0.04 1x 438 5.69 0.57 1.59 0.26 1x 317 4.61 0.09 1.25 0.13 1x 303 5.41 0.11 1.52 0.15 1x 310 6.65 0.06 1.93 0.19 4x 438 1.68 0.24 0.50 0.03 4x 412 1.29 0.14 0.35 0.01 6x 415 0.50 0.15 0.14 0.03 5x 317 5.15 0.28 1.43 0.07 5x 303 0.63 0.07 0.19 0.03 5x 310 0.52 0.07 0.15 0.03 4x 159 24.81 2.38 7.72 0.82 4x 295 4.92 0.56 1.60 0.26 4x 283 18.70 0.58 5.87 0.57 4x 301 0.45 0.06 0.14 0.01 4x 410 1.49 0.05 0.47 0.05 4x 451 2.13 0.12 0.68 0.05 4x 161 1.80 0.14 0.57 0.03 RMs126 (1x 14.21 1.11 4.56 0.63 pGAP 18B) RMs127 (3x 28.61 2.05 8.81 0.80 pGAP 18B) RMs134 (4x 30.89 1.48 9.73 0.83 pGAP 18B) RMs133 (1x pGCW14 18B) 36.90 2.43 12.14 1.39 RMs138 (2x pGCW14 18B) 47.31 3.66 16.42 1.45 RMs143 (3x pGCW14 18B) 56.49 0.97 20.96 0.72 RMs152 (4x pGCW14 18B) 58.06 4.31 20.97 3.74 RM411 (6B, un-rotated) 12.01 1.16 3.76 0.31 RM434 (12B, un-rotated) 17.57 1.47 5.50 0.22 RM439 (18B, un-rotated) 14.36 1.25 4.56 0.21 RM2361 (30B, un-rotated) 8.81 0.58 2.87 0.39

To measure productivity, 3 clones of each strain were inoculated into 400 μl of Buffered Glycerol-complex Medium (BMGY) in a 96-well square-well block, and incubated for 48 hours at 30° C. with agitation at 1000 rpm. Following the 48-hour incubation, 4 μl of each culture was used to inoculate a fresh 400 μl of BMGY in a 96-well square-well block, which was then incubated for 24 hours 30° C. with agitation at 1000 rpm. Cells were then pelleted by centrifugation, the supernatant removed, and the cells resuspended in 400 μl of fresh BMGY. The cells were again pelleted by centrifugation, the supernatant removed, and the cells resuspended in 800 μl of fresh BMGY. From that 800 μl, 400 μl was aliquoted into a 96-well square-well block, which was then incubated for 2 hours at 30° C. with agitation at 1000 rpm. After the 2 hours, the OD600 of the cultures was recorded, and the cells were pelleted by centrifugation and the supernatant collected for further analysis. The concentration of block copolymer polypeptide in each supernatant was determined by direct enzyme-linked immunosorbent assay (ELISA) analysis quantifying the 3×FLAG epitope.

The relative productivities of each strain confirm qualitative observations made based on western blot data. The circularly permuted polypeptides express at similar levels to un-rotated silks, stronger promoters or more copies lead to higher block copolymer expression and secretion, and longer block copolymer polypeptides comprising silk repeat sequences generally express less well than shorter block copolymers comprising the same or different repeat sequences. Interestingly, the grams of 12B (55.9 kDa) produced exceeds the grams of 6B (29.5 kDa) produced, suggesting that the factors leading to decreased expression of larger block copolymers comprising silk repeat sequences may not become dominant until expression of block copolymers closer to the size of 18B (82.2 kDa) Importantly, most of the block copolymer polypeptides have a relatively high specific productivity (>0.1 mg silk/g Dry Cell Weight (DCW)/hour. In some embodiments, the productivity is above 2 mg silk/g DCW/hour. In further embodiments, the productivity is above 5 mg silk/g DCW/hour), before any optimization of the level of polypeptide transcription. Additional transcription improved the productivity of 18B by approximately 5-fold to 20 (almost 21) mg polypeptide/g DCW/hour.

Example 14 Measuring Mechanical Properties of Silk Fiber

The block copolymer polypeptide produced in Example 5 was spun into a fiber and tested for various mechanical properties. First, a fiber spinning solution was prepared by dissolving the purified and dried block copolymer polypeptide in a formic acid-based spinning solvent, using standard techniques. Spin dopes were incubated at 35° C. on a rotational shaker for three days with occasional mixing. After three days, the spin dopes were centrifuged at 16000 rcf for 60 minutes and allowed to equilibrate to room temperature for at least two hours prior to spinning.

The spin dope was extruded through a 50-200 μm diameter orifice into a standard alcohol-based coagulation bath. Fibers were pulled out of the coagulation bath under tension, drawn from 1 to 5 times their length, and subsequently allowed to dry. At least five fibers were randomly selected from the at least 10 meters of spun fibers. These fibers were tested for tensile mechanical properties using an instrument including a linear actuator and calibrated load cell. Fibers were pulled at 1% strain until failure. Fiber diameters were measured with light microscopy at 20× magnification using image processing software. The mean maximum stress ranged from 54-310 MPa. The mean yield stress ranged from 24-172 MPa. The mean maximum strain ranged from 2-200%. Th mean initial modulus ranged from 1617-5820 MPa. The effect of the draw ratio is illustrated in Table 12 and FIG. 17. Also, the average toughness of three fibers was measured at 0.5 MJ m⁻³ (standard deviation of 0.2), 20 MJ m⁻³ (standard deviation of 0.9), and 59.2 MJ m³ (standard deviation of 8.9)

TABLE 12 Effect of draw ratio 2.5x 5x Mean Maximum Stress (MPa) 58 80 Mean Yield Stress (Mpa) 53 61 Mean max strain (%) 277 94 Mean initial modulus (MPa) 1644 2719

Fiber diameters were determined as the average of at least 4-8 fibers selected randomly from at least 10 m of spun fibers. For each fiber, six measurements were made over the span of 0.57 cm. The diameters ranged from 4.48-12.7 μm. Fiber diameters were consistent within the same sample. Samples ranged over various average diameters: 10.3 μm (standard deviation of 0.4 μm), 13.47 μm (standard deviation of 0.36 μm), 12.05 μm (standard deviation of 0.67), 14.69 μm (standard deviation of 0.76 μm), and 9.85 μm (standard deviation of 0.38 μm).

One particularly effective fiber which was spun from block copolymer material that was generated from an optimized recovery and separations protocol had a maximum ultimate tensile strength of 310 MPa, a mean diameter of 4.9 μm (standard deviation of 0.8), and a max strain of 20%. Fiber tensile test results are shown in FIG. 18.

Fibers were dried overnight at room temperature. FTIR spectra were collected with a diamond ATR module from 400 cm⁻¹ to 4000 cm⁻¹ with 4 cm⁻¹ resolution (FIG. 19). The amide I region (1600 cm⁻¹ to 1700 cm⁻¹) was baselined and curve fitted with Gaussian profiles at 5-6 location determined by peak locations from the second derivative of the original curve. The β-sheet content was determined as the area under the Gaussian profile at ˜1620 cm⁻¹ and ˜1690 cm⁻¹ divided by the total area of the amide I region. Annealed and untreated fibers were tested. For annealing, fibers were incubated within a humidified vacuum chamber at 1.5 Torr for at least six hours. Untreated fibers were found to contain 31% β-sheet content, and annealed fibers were found to contain 50% β-sheet content.

Fiber cross-sections were examined by freeze fracture using liquid nitrogen. Samples were sputter coated with platinum/palladium and imaged with a Hitachi TM-1000 at 5 kV accelerating voltage. FIG. 20 shows that the fibers have smooth surfaces, circular cross sections, and are solid and free of voids. In some embodiments

Example 15 Production of Optimal Fibers

An R domain of MaSp2-like silks is selected from those listed in Tables 13a and 13b, and the R domain is concatenated into 4× repeat domains flanked by alpha mating factor on the 5′ end and 3× FLAG on the 3′ end using the assembly scheme shown in FIG. 12. The concatenation is performed as described in Example 4 and shown in FIG. 7 and FIG. 8. The resulting polynucleotide sequence and corresponding polypeptide sequences are listed in Tables 13a and 13b.

Of the sequences in Tables 13a and 13b: (1) the proline content ranges from 11.35-15.74% (the percentages of Tables 13a and 13b refer to a number of amino acid residues of the specified content—in this case, proline—over a total number of amino acid residues in the corresponding polypeptide sequence). The proline content of similar R domains could also range between 13-15%, 11-16%, 9-20%, or 3-24%; (2) the alanine content ranges between 16.09-30.51%. The alanine content of similar R domains could also range between 15-20%, 16-31%, 12-40%, or 8-49%; (3) the glycine content ranges between 29.66-42.15%. The glycine content of similar R domains could also range between 38-43%, 29-43%, 25-50%, or 21-57%; (4) The glycine and alanine content ranges between 54.17-68.59%. The glycine and alanine content of similar R domains could also range between 54-69%, 48-75%, or 42-81%; (5) the β-turn content ranges between 18.22-32.16%. β-turn content is calculated using the SOPMA method from Geourjon, C., and Deleage, G., SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments, Comput. Appl. Biosci., 11:6, pg. 681-684 (1995). The SOPMA method is applied using the following parameters: window width—10; similarity threshold—10; number of states—4. The β-turn content of similar R domains could also range between 25-30%, 18-33%, 15-37%, or 12-41%; (6) the poly-alanine content ranges between 12.64-28.85%. A motif is considered a poly-alanine motif if it includes at least four consecutive alanine residues. The poly-alanine content of similar R domains could also range between 12-29%, 9-35%, or 6-41%; (7) the GPG motif content ranges between 22.95-46.67%. The GPG motif content of similar R domains could also range between 30-45%, 22-47%, 18-55%, or 14-63%; (8) the GPG and poly-alanine content ranges between 42.21-73.33%. The GPG and poly-alanine content of similar R domains could also range between 25-50%, 20-60%, or 15-70%. Other silk types exhibit different ranges of amino acid content and other properties. FIG. 21 shows ranges of glycine, alanine, and proline content for various silk types of the silk polypeptide sequences disclosed herein. FIG. 21 illustrates percentages of glycine, alanine, or proline amino acid residues over a total number of residues in the polypeptide sequences.

The resulting product of the concatenation comprising 4 repeat sequences, an alpha mating factor, and a 3× FLAG domain is digested with AscI and SbfI to release the desired silk sequence and ligated into expression vectors RM812 (SEQ ID N: 2701), RM837 (SEQ ID NO: 2702), RM814 (SEQ ID NO: 2703), and RM815 (SEQ ID NO: 2704) (key attributes of the expression vectors are summarized in Table 9) that have been digested with AscI and SbfI. A strain containing 4 copies of the silk polynucleotide under the transcriptional control of pGCW14 is generated by serially transforming Pichia (Komagataella) pastoris strain GS115 (NRRL Y15851) with the resulting expression vectors (after linearizing them with BsaI) using the PEG method. Similar quasi-repeat domains can range between 500-5000, 119-1575, 300-1200, 500-1000, or 900-950 amino acids in length. The entire block co-polymer can range between 40-400, 12.2-132, 50-200, or 70-100 kDa.

TABLE 13a Properties of selected R domains Alpha Mating Alpha Mating Factor + Factor + 4x Repeat 4x Repeat 1x Repeat 1x Repeat Domain + Domain + Domain Domain 3xFLAG 3xFLAG % Amino Acid DNA Amino Acid DNA % % % Glycine + SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO Proline Alanine Glycine Alanine 1313 382 2752 2777 14.22 21.10 38.07 59.17 1314 383 2753 2778 14.75 20.86 37.77 58.63 1315 384 2754 2779 14.74 18.33 39.84 58.17 1316 385 2755 2780 14.91 18.42 39.91 58.33 1317 386 2756 2781 14.79 18.68 39.69 58.37 1318 387 2757 2782 14.12 19.22 40.78 60.00 1319 388 2758 2783 14.68 18.65 39.68 58.33 1320 389 2759 2784 14.56 16.09 42.15 58.24 1321 390 2760 2785 14.73 18.99 39.53 58.53 1328 397 2761 2786 15.00 20.71 38.57 59.29 1329 398 2762 2787 14.29 20.71 38.57 59.29 1331 400 2763 2788 14.39 20.14 38.13 58.27 1335 404 2764 2789 11.86 30.51 29.66 60.17 1336 405 2765 2790 12.72 24.12 35.96 60.09 1337 406 2766 2791 13.52 22.54 35.25 57.79 1340 409 2767 2792 11.35 20.09 37.99 58.08 1370 439 2768 2793 15.74 17.13 37.04 54.17 1373 442 2769 2794 15.56 26.67 40.00 66.67 1374 443 2770 2795 14.22 28.89 38.22 67.11 1375 444 2771 2796 14.35 26.85 39.35 66.20 1376 445 2772 2797 15.18 26.79 39.29 66.07 1378 447 2773 2798 14.44 27.81 39.04 66.84 1379 448 2774 2799 14.94 25.86 40.80 66.67 1380 449 2775 2800 14.10 29.49 39.10 68.59 1384 453 2776 2801 12.16 25.00 35.81 60.81

TABLE 13b Properties of selected R domains Alpha Mating Alpha Mating Factor + Factor + 4x Repeat 4x Repeat 1x Repeat 1x Repeat Domain + Domain + % Domain Domain 3xFLAG 3xFLAG % % % GPG + Amino Acid DNA Amino Acid DNA Beta Poly GPG Poly SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO Turn alanine motif Alanine MW 1313 382 2752 2777 28.44 17.89 27.52 45.41 76044 1314 383 2753 2778 30.22 17.63 28.06 45.68 95860 1315 384 2754 2779 30.68 15.54 32.27 47.81 86818 1316 385 2755 2780 28.51 14.91 31.58 46.49 79731 1317 386 2756 2781 28.79 15.56 32.68 48.25 89297 1318 387 2757 2782 32.16 16.08 30.59 46.67 88136 1319 388 2758 2783 30.56 15.87 32.14 48.02 87103 1320 389 2759 2784 28.74 12.64 31.03 43.68 90778 1321 390 2760 2785 28.68 15.89 32.56 48.45 89582 1328 397 2761 2786 31.43 17.86 32.14 50.00 49712 1329 398 2762 2787 29.29 17.86 30.00 47.86 49836 1331 400 2763 2788 29.50 17.27 30.22 47.48 49672 1335 404 2764 2789 18.22 24.58 25.42 50.00 83965 1336 405 2765 2790 25.00 19.74 30.26 50.00 80845 1337 406 2766 2791 22.54 18.85 22.95 42.21 87160 1340 409 2767 2792 20.09 16.59 27.51 44.10 81149 1370 439 2768 2793 26.85 15.28 40.28 55.56 77581 1373 442 2769 2794 25.78 26.67 46.67 73.33 76502 1374 443 2770 2795 26.67 28.00 42.67 70.67 75716 1375 444 2771 2796 24.07 26.39 43.06 69.44 73742 1376 445 2772 2797 28.12 26.34 44.20 70.54 76433 1378 447 2773 2798 24.60 27.27 43.32 70.59 63684 1379 448 2774 2799 25.86 25.86 44.83 70.69 59391 1380 449 2775 2800 27.56 28.85 42.31 71.15 53049 1384 453 2776 2801 28.38 18.24 24.32 42.57 52668

A clone of the resulting strain is cultured according to the following conditions: the culture is grown in a minimal basal salt media, similar to one described in [http://tools.invitrogen.com/content/sfs/manuals/pichiaferm_prot.pdf] with 50 g/L of glycerol as a starting feedstock. Growth occurs in a stirred fermentation vessel controlled at 30 C, with 1 VVM of air flow and 2000 rpm agitation. pH is controlled at 3 with the on-demand addition of ammonium hydroxide. Additional glycerol is added as needed based on sudden increases in dissolved oxygen. Growth is allowed to continue until dissolved oxygen reached 15% of maximum at which time the culture is harvested, typically at 200-300 OD of cell density.

The broth from the fermenter is decellularized by centrifugation. The supernatant from the Pichia (Komagataella) pastoris culture is collected. Low molecular weight components are removed from the supernatant using ultrafiltration to remove particles smaller than the block copolymer polypeptides. The filtered culture supernatant is then concentrated up to 50×.

The fiber spinning solution is prepared by dissolving the purified and dried block copolymer polypeptide in a formic acid-based spinning solvent. Spin dopes are incubated at 35° C. on a rotational shaker for three days with occasional mixing. After three days, the spin dopes are centrifuged at 16000 rcf for 60 minutes and allowed to equilibrate to room temperature for at least two hours prior to spinning. The spin dope is extruded through a 150 μm diameter orifice into a standard alcohol-based coagulation bath. Fibers are pulled out of the coagulation bath under tension, drawn from 1 to 5 times their length, and subsequently allowed to dry as a tight hank.

At least five fibers are randomly selected from at least 10 meters of spun fibers. Fibers are tested for tensile mechanical properties using a custom instrument, which includes a linear actuator and calibrated load cell. Fibers are mounted with a gauge length of 5.75 mm and pulled at a 1% strain rate until failure. The ultimate tensile strengths of the fibers are measured to be between 50-500 MPa. Depending on which fibers are selected: the yield stress is measured to be 24-172 MPa or 150-172 MPa, the ultimate tensile strength (maximum stress) is measured to be 54-310 MPa or 150-310 MPa, the breaking strain is measured to be 2-200% or 180-200%, the initial modulus is measured to be 1617-5820 MPa or 5500-5820 MPa, and the toughness value is measured to be at least 0.5 MJ/m³, at least 3.1 MJ/m³, or at least 59.2 MJ/m³.

The resultant forces are normalized to the fiber diameter, as measured by light microscopy. Fiber diameters are measured with light microscopy at 20× magnification using image processing software. Fiber diameters are determined as the average of at least 4-8 fibers selected randomly from at least 10 m of spun fibers. For each fiber, six measurements are made over the span of 5.75 mm Depending on which fibers are selected, the fiber diameters are measured to be between 4-100 μm, between 4.48-12.7 μm, or between 4-5 μm.

To test the β-sheet crystallinity content of the fibers, the fibers are dried overnight at room temperature. FTIR spectra are collected with a diamond ATR module from 400 cm⁻¹ to 4000 cm⁻¹ with 4 cm⁻¹ resolution. The amide I region (1600 cm⁻¹ to 1700 cm⁻¹) is baselined and curve fitted with Gaussian profiles at 5-6 location determined by peak locations from the second derivative of the original curve. The β-sheet content is determined as the area under the Gaussian profile at ˜1620 cm⁻¹ and ˜1690 cm⁻¹ divided by the total area of the amide I region. To induce β-sheet crystallinity, fibers are incubated within a humidified vacuum chamber at 1.5 Torr for at least six hours. Fiber surface morphology and cross-sections (taken by freeze fracture using liquid nitrogen) are analyzed via scanning electron microscopy. Samples are sputter coated with platinum/palladium and imaged with a Hitachi TM-1000 at 5 kV accelerating voltage.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. 

What is claimed is:
 1. A proteinaceous block co-polymer, comprising: a polypeptide, the polypeptide comprising from 2 to 8 concatenated repeats of SEQ ID NO: 1396 or circularly permuted variants thereof.
 2. The proteinaceous block co-polymer of claim 1, wherein the polypeptide comprises a property selected from the group consisting of an alanine composition from 12 to 40%, a glycine composition from 25 to 50%, a proline composition from 9 to 20%, a β-turn composition from 15 to 37%, a GPG amino acid motif content from 18 to 55%, and a poly-alanine amino acid motif content from 9 to 35%.
 3. The proteinaceous block co-polymer of claim 1, consisting of: 3 concatenated repeats of SEQ ID NO:
 1396. 4. A fiber comprising the block co-polymer of claim
 1. 5. The fiber of claim 4, comprising a circular cross-section and a diameter from 4.48 to 12.7 μm.
 6. The fiber of claim 4, wherein the fiber has a yield stress from 24 to 172 MPa.
 7. The fiber of claim 4, wherein the fiber has a maximum stress from 54 to 310 MPa.
 8. The fiber of claim 4, wherein the fiber has a breaking strain from 2 to 200%.
 9. The fiber of claim 4, wherein the fiber has an initial modulus from 1617 to 5820 MPa.
 10. The fiber of claim 4, wherein the fiber has a toughness value from 0.5 MJ/m³ to 59.2 MJ/m³.
 11. An expression construct encoding the proteinaceous block co-polymer of claim
 1. 12. The expression construct of claim 11, further comprising a secretion signal operatively linked to the coding sequence for the block copolymer polypeptide.
 13. The expression construct of claim 12, wherein the secretion signal comprises an alpha mating factor leader and pro sequence.
 14. A host cell, comprising: the expression construct of claim
 11. 15. A host cell, comprising: the expression construct of claim
 12. 16. A host cell, comprising: the expression construct of claim
 13. 17. A method of producing the proteinaceous block copolymer of claim 1, comprising: culturing the host cell of claim 14 under conditions that promote expression of the block copolymer polypeptide from the expression construct; and recovering the expressed block copolymer.
 18. A method of producing the proteinaceous block copolymer of claim 1, comprising: culturing the host cell of claim 15 under conditions that promote expression of the block copolymer polypeptide from the expression construct; and recovering the secreted block copolymer.
 19. A culture medium comprising a secreted block copolymer of claim
 1. 20. A method for producing a fiber, comprising: obtaining the cell culture medium of claim 19; isolating the secreted protein; processing the protein into a spinnable solution; and producing a fiber from the spinnable solution. 